Preclinical evaluation of therapeutic vaccines for chronic hepatitis B that stimulate antiviral activities of T cells and NKT cells.

Background & Aims: Liver diseases resulting from chronic HBV infection are a significant cause of morbidity and mortality. Vaccines that elicit T-cell responses capable of controlling the virus represent a treatment strategy with potential for long-term effects. Here, we evaluated vaccines that induce the activity of type I natural killer T (NKT) cells to limit viral replication and license stimulation of conventional antiviral T-cells. Methods: Vaccines were prepared by conjugating peptide epitopes to an NKT-cell agonist to promote co-delivery to antigen-presenting cells, encouraging NKT-cell licensing and stimulation of T cells. Activity of the conjugate vaccines was assessed in transgenic mice expressing the complete HBV genome, administered intravenously to maximise access to NKT cell-rich tissues. Results: The vaccines induced only limited antiviral activity in unmanipulated transgenic hosts, likely attributable to NKT-cell activation as T-cell tolerance to viral antigens is strong. However, in a model of chronic hepatitis B involving transfer of naive HBcAg-specific CD8+ T cells into the transgenic mice, which typically results in specific T-cell dysfunction without virus control, vaccines containing the targeted HBcAg epitope induced prolonged antiviral activity because of qualitatively improved T-cell stimulation. In a step towards a clinical product, vaccines were prepared using synthetic long peptides covering clusters of known HLA-binding epitopes and shown to be immunogenic in HLA transgenic mice. Predictions based on HLA distribution suggest a product containing three selected SLP-based vaccines could give >90 % worldwide coverage, with an average of 3.38 epitopes targeted per individual. Conclusions: The novel vaccines described show promise for further clinical development as a treatment for chronic hepatitis B. Impact and Implications: Although there are effective prophylactic vaccines for HBV infection, it is estimated that 350-400 million people worldwide have chronic hepatitis B, putting these individuals at significant risk of life-threatening liver diseases. Therapeutic vaccination aimed at activating or boosting HBV-specific T-cell responses holds potential as a strategy for treating chronic infection, but has so far met with limited success. Here, we show that a glycolipid-peptide conjugate vaccine designed to coordinate activity of type I NKT cells alongside conventional antiviral T cells has antiviral activity in a mouse model of chronic infection. It is anticipated that a product based on a combination of three such conjugates, each prepared using long peptides covering clusters of known HLA-binding epitopes, could be developed further as a treatment for chronic hepatitis B with broad global HLA coverage.

Highlights from the 2023 International Meeting on the Molecular Biology of Hepatitis B virus.

Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.

Antibody-independent protection against heterologous SARS-CoV-2 challenge conferred by prior infection or vaccination.

Vaccines have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity and mortality, yet emerging variants challenge their effectiveness. The prevailing approach to updating vaccines targets the antibody response, operating under the presumption that it is the primary defense mechanism following vaccination or infection. This perspective, however, can overlook the role of T cells, particularly when antibody levels are low or absent. Here we show, through studies in mouse models lacking antibodies but maintaining functional B cells and lymphoid organs, that immunity conferred by prior infection or mRNA vaccination can protect against SARS-CoV-2 challenge independently of antibodies. Our findings, using three distinct models inclusive of a novel human/mouse ACE2 hybrid, highlight that CD8+ T cells are essential for combating severe infections, whereas CD4+ T cells contribute to managing milder cases, with interferon-γ having an important function in this antibody-independent defense. These findings highlight the importance of T cell responses in vaccine development, urging a broader perspective on protective immunity beyond just antibodies.

CD8 cis-targeted IL-2 drives potent antiviral activity against hepatitis B virus.

CD8+ T cells are key antiviral effectors against hepatitis B virus (HBV), yet their number and function can be compromised in chronic infections. Preclinical HBV models displaying CD8+ T cell dysfunction showed that interleukin-2 (IL-2)-based treatment, unlike programmed cell death ligand 1 (PD-L1) checkpoint blockade, could reverse this defect, suggesting its therapeutic potential against HBV. However, IL-2's effectiveness is hindered by its pleiotropic nature, because its receptor is found on various immune cells, including regulatory T (Treg) cells and natural killer (NK) cells, which can counteract antiviral responses or contribute to toxicity, respectively. To address this, we developed a cis-targeted CD8-IL2 fusion protein, aiming to selectively stimulate dysfunctional CD8+ T cells in chronic HBV. In a mouse model, CD8-IL2 boosted the number of HBV-reactive CD8+ T cells in the liver without substantially altering Treg or NK cell counts. These expanded CD8+ T cells exhibited increased interferon-γ and granzyme B production, demonstrating enhanced functionality. CD8-IL2 treatment resulted in substantial antiviral effects, evidenced by marked reductions in viremia and antigenemia and HBV core antigen-positive hepatocytes. In contrast, an untargeted CTRL-IL2 led to predominant NK cell expansion, minimal CD8+ T cell expansion, negligible changes in effector molecules, and minimal antiviral activity. Human CD8-IL2 trials in cynomolgus monkeys mirrored these results, achieving a roughly 20-fold increase in peripheral blood CD8+ T cells without affecting NK or Treg cell numbers. These data support the development of CD8-IL2 as a therapy for chronic HBV infection.

The interplay of drug therapeutics and immune responses to SARS-CoV-2.

The SARS-CoV-2 pandemic has necessitated rapid therapeutic and preventative responses. While vaccines form the frontline of defense, antiviral treatments such as nirmatrelvir have emerged as vital adjunctive measures, particularly for those unable or unwilling to be vaccinated. This review delves into the potential influence of nirmatrelvir on enduring immunity. In parallel, the potential of drug repurposing is explored, with bisphosphonates being examined for their possible effects against COVID-19 due to their immunomodulatory properties. The importance of rigorous clinical trials and careful interpretation of preliminary data is emphasized.

Inhibition of the lysine demethylase LSD1 modulates the balance between inflammatory and antiviral responses against coronaviruses.

Innate immune responses to coronavirus infections are highly cell specific. Tissue-resident macrophages, which are infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients but are inconsistently infected in vitro, exert critical but conflicting effects by secreting both antiviral type I interferons (IFNs) and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for the treatment of coronavirus disease 2019 (COVID-19), indiscriminately suppress both responses, possibly impairing viral clearance. Here, we established in vitro cell culture systems that enabled us to separately investigate the cell-intrinsic and cell-extrinsic proinflammatory and antiviral activities of mouse macrophages infected with the prototypical murine coronavirus MHV-A59. We showed that the nuclear factor κB-dependent inflammatory response to viral infection was selectively inhibited by loss of the lysine demethylase LSD1, which was previously implicated in innate immune responses to cancer, with negligible effects on the antiviral IFN response. LSD1 ablation also enhanced an IFN-independent antiviral response, blocking viral egress through the lysosomal pathway. The macrophage-intrinsic antiviral and anti-inflammatory activity of Lsd1 inhibition was confirmed in vitro and in a humanized mouse model of SARS-CoV-2 infection. These results suggest that LSD1 controls innate immune responses against coronaviruses at multiple levels and provide a mechanistic rationale for potentially repurposing LSD1 inhibitors for COVID-19 treatment.

Immunogenicity and Pre-Clinical Efficacy of an OMV-Based SARS-CoV-2 Vaccine.

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespective of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccines to induce potent immune responses against the associated proteins. Here, we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in vaccinated mice, resulting in the production of neutralizing antibodies (nAbs) with a titre higher than 1:300. The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induce nAbs against Omicron BA.1 and BA.5, as measured using the pseudovirus neutralization infectivity assay. Importantly, we show that the RBM438-509 ancestral-OMVs elicited antibodies which efficiently neutralize in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants with a neutralization titre ranging from 1:100 to 1:1500, suggesting its potential use as a vaccine targeting diverse SARS-CoV-2 variants. Altogether, given the convenience associated with the ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

A fluorescent reporter model for the visualization and characterization of TDC.

TDC are hematopoietic cells that combine dendritic cell (DC) and conventional T-cell markers and functional properties. They were identified in secondary lymphoid organs (SLOs) of naïve mice as cells expressing CD11c, major histocompatibility molecules (MHC)-II, and the T-cell receptor (TCR). Despite thorough characterization, a physiological role for TDC remains to be determined. Unfortunately, using CD11c as a marker for TDC has the caveat of its upregulation on different cells, including T cells, upon activation. Here, we took advantage of Zbtb46-GFP reporter mice to explore the frequency and localization of TDC in different tissues at steady state and upon viral infection. RNA sequencing analysis confirmed that TDC sorted from Zbtb46-GFP mice have a gene signature that is distinct from conventional T cells and DC. In addition, this reporter model allowed for identification of TDC in situ not only in SLOs but also in the liver and lung of naïve mice. Interestingly, we found that TDC numbers in the SLOs increased upon viral infection, suggesting that TDC might play a role during viral infections. In conclusion, we propose a visualization strategy that might shed light on the physiological role of TDC in several pathological contexts, including infection and cancer.

Immunogenicity and pre-clinical efficacy of an OMV-based SARS-CoV-2 vaccine.

The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespectively of economical and climatic conditions. Outer membrane vesicles (OMV) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMV can be used as vaccine to induce potent immune responses against the associated protein. Here we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in vaccinated mice, resulting in the production of neutralizing antibodies (nAbs). The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induced nAbs against Omicron BA.1 and BA.5, as judged by pseudovirus infectivity assay. Importantly, we show that the RBM438-509 ancestral-OMVs elicited antibodies which efficiently neutralized in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants, suggesting its potential use as a pan SARS-CoV-2 vaccine. Altogether, given the convenience associated with ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.

SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the RNA virus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Although SARS-CoV-2 was reported to alter several cellular pathways, its impact on DNA integrity and the mechanisms involved remain unknown. Here we show that SARS-CoV-2 causes DNA damage and elicits an altered DNA damage response. Mechanistically, SARS-CoV-2 proteins ORF6 and NSP13 cause degradation of the DNA damage response kinase CHK1 through proteasome and autophagy, respectively. CHK1 loss leads to deoxynucleoside triphosphate (dNTP) shortage, causing impaired S-phase progression, DNA damage, pro-inflammatory pathways activation and cellular senescence. Supplementation of deoxynucleosides reduces that. Furthermore, SARS-CoV-2 N-protein impairs 53BP1 focal recruitment by interfering with damage-induced long non-coding RNAs, thus reducing DNA repair. Key observations are recapitulated in SARS-CoV-2-infected mice and patients with COVID-19. We propose that SARS-CoV-2, by boosting ribonucleoside triphosphate levels to promote its replication at the expense of dNTPs and by hijacking damage-induced long non-coding RNAs' biology, threatens genome integrity and causes altered DNA damage response activation, induction of inflammation and cellular senescence.

Nirmatrelvir treatment of SARS-CoV-2-infected mice blunts antiviral adaptive immune responses.

Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS-CoV-2 pandemic. Nirmatrelvir-an orally available inhibitor of the 3-chymotrypsin-like cysteine protease-has been shown to reduce the risk of progression to severe COVID-19. However, the impact of nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses is unknown. Here, by using mouse models of SARS-CoV-2 infection, we show that nirmatrelvir administration blunts the development of SARS-CoV-2-specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir-treated mice recruited significantly fewer memory T and B cells to the infected lungs and mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.

Pyrimidine de novo synthesis inhibition selectively blocks effector but not memory T cell development.

Blocking pyrimidine de novo synthesis by inhibiting dihydroorotate dehydrogenase is used to treat autoimmunity and prevent expansion of rapidly dividing cell populations including activated T cells. Here we show memory T cell precursors are resistant to pyrimidine starvation. Although the treatment effectively blocked effector T cells, the number, function and transcriptional profile of memory T cells and their precursors were unaffected. This effect occurred in a narrow time window in the early T cell expansion phase when developing effector, but not memory precursor, T cells are vulnerable to pyrimidine starvation. This vulnerability stems from a higher proliferative rate of early effector T cells as well as lower pyrimidine synthesis capacity when compared with memory precursors. This differential sensitivity is a drug-targetable checkpoint that efficiently diminishes effector T cells without affecting the memory compartment. This cell fate checkpoint might therefore lead to new methods to safely manipulate effector T cell responses.

Group 1 ILCs regulate T cell-mediated liver immunopathology by controlling local IL-2 availability.

Group 1 innate lymphoid cells (ILCs), which comprise both natural killer (NK) cells and ILC1s, are important innate effectors that can also positively and negatively influence adaptive immune responses. The latter function is generally ascribed to the ability of NK cells to recognize and kill activated T cells. Here, we used multiphoton intravital microscopy in mouse models of hepatitis B to study the intrahepatic behavior of group 1 ILCs and their cross-talk with hepatitis B virus (HBV)-specific CD8+ T cells. We found that hepatocellular antigen recognition by effector CD8+ T cells triggered a prominent increase in the number of hepatic NK cells and ILC1s. Group 1 ILCs colocalized and engaged in prolonged interactions with effector CD8+ T cells undergoing hepatocellular antigen recognition; however, they did not induce T cell apoptosis. Rather, group 1 ILCs constrained CD8+ T cell proliferation by controlling local interleukin-2 (IL-2) availability. Accordingly, group 1 ILC depletion, or genetic removal of their IL-2 receptor a chain, considerably increased the number of intrahepatic HBV-specific effector CD8+ T cells and the attendant immunopathology. Together, these results reveal a role for group 1 ILCs in controlling T cell-mediated liver immunopathology by limiting local IL-2 concentration and have implications for the treatment of chronic HBV infection.

Protective immune trajectories in early viral containment of non-pneumonic SARS-CoV-2 infection.

The antiviral immune response to SARS-CoV-2 infection can limit viral spread and prevent development of pneumonic COVID-19. However, the protective immunological response associated with successful viral containment in the upper airways remains unclear. Here, we combine a multi-omics approach with longitudinal sampling to reveal temporally resolved protective immune signatures in non-pneumonic and ambulatory SARS-CoV-2 infected patients and associate specific immune trajectories with upper airway viral containment. We see a distinct systemic rather than local immune state associated with viral containment, characterized by interferon stimulated gene (ISG) upregulation across circulating immune cell subsets in non-pneumonic SARS-CoV2 infection. We report reduced cytotoxic potential of Natural Killer (NK) and T cells, and an immune-modulatory monocyte phenotype associated with protective immunity in COVID-19. Together, we show protective immune trajectories in SARS-CoV2 infection, which have important implications for patient prognosis and the development of immunomodulatory therapies.

COVID-eVax, an electroporated DNA vaccine candidate encoding the SARS-CoV-2 RBD, elicits protective responses in animal models.

The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.

Administration of aerosolized SARS-CoV-2 to K18-hACE2 mice uncouples respiratory infection from fatal neuroinvasion.

The development of a tractable small animal model faithfully reproducing human coronavirus disease 2019 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high central nervous system infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model's usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction but did not lead to fatal viral neuroinvasion. When compared with intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition, and a transcriptional signature comparable to that observed in SARS-CoV-2­infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection), and therapeutic interventions.

Isolation of mouse Kupffer cells for phenotypic and functional studies.

Here, we provide detailed protocols for the isolation of mouse Kupffer cells - the liver-resident macrophages - for phenotypic (e.g., via flow cytometry, mass cytometry, or RNA-sequencing) analyses or for functional experiments involving cell culture. The procedures presented can be adapted for the isolation of other hepatic cell populations. For complete details on the use and execution of this protocol, please refer to De Simone et al. (2021).

A subset of Kupffer cells regulates metabolism through the expression of CD36.

Tissue macrophages are immune cells whose phenotypes and functions are dictated by origin and niches. However, tissues are complex environments, and macrophage heterogeneity within the same organ has been overlooked so far. Here, we used high-dimensional approaches to characterize macrophage populations in the murine liver. We identified two distinct populations among embryonically derived Kupffer cells (KCs) sharing a core signature while differentially expressing numerous genes and proteins: a major CD206loESAM- population (KC1) and a minor CD206hiESAM+ population (KC2). KC2 expressed genes involved in metabolic processes, including fatty acid metabolism both in steady-state and in diet-induced obesity and hepatic steatosis. Functional characterization by depletion of KC2 or targeted silencing of the fatty acid transporter Cd36 highlighted a crucial contribution of KC2 in the liver oxidative stress associated with obesity. In summary, our study reveals that KCs are more heterogeneous than anticipated, notably describing a subpopulation wired with metabolic functions.

Identification of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming.

Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages known for their scavenger and phagocytic functions. KCs can also present antigens to CD8+ T cells and promote either tolerance or effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection, in which HBV-specific naive CD8+ T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction, to identify a subset of KCs (referred to as KC2) that cross-presents hepatocellular antigens upon interleukin-2 (IL-2) administration, thus improving the antiviral function of T cells. Removing MHC-I from all KCs, including KC2, or selectively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. In summary, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment, suggesting new strategies for boosting hepatic T cell immunity.

Serum HBsAg clearance has minimal impact on CD8+ T cell responses in mouse models of HBV infection.

Antibody-mediated clearance of hepatitis B surface antigen (HBsAg) from the circulation of chronically infected patients (i.e., seroconversion) is usually associated with increased HBV-specific T cell responsiveness. However, a causative link between serum HBsAg levels and impairment of intrahepatic CD8+ T cells has not been established. Here we addressed this issue by using HBV replication-competent transgenic mice that are depleted of circulating HBsAg, via either spontaneous seroconversion or therapeutic monoclonal antibodies, as recipients of HBV-specific CD8+ T cells. Surprisingly, we found that serum HBsAg clearance has only a minimal effect on the expansion of HBV-specific naive CD8+ T cells undergoing intrahepatic priming. It does not alter their propensity to become dysfunctional, nor does it enhance the capacity of IL-2-based immunotherapeutic strategies to increase their antiviral function. In summary, our results reveal that circulating HBsAg clearance does not improve HBV-specific CD8+ T cell responses in vivo and may have important implications for the treatment of chronic HBV infection.