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.

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.

Priming and Maintenance of Adaptive Immunity in the Liver.

The liver's unique characteristics have a profound impact on the priming and maintenance of adaptive immunity. This review delves into the cellular circuits that regulate adaptive immune responses in the liver, with a specific focus on hepatitis B virus infection as an illustrative example. A key aspect highlighted is the liver's specialized role in priming CD8+ T cells, leading to a distinct state of immune hyporesponsiveness. Additionally, the influence of the liver's hemodynamics and anatomical features, particularly during liver fibrosis and cirrhosis, on the differentiation and function of adaptive immune cells is discussed. While the primary emphasis is on CD8+ T cells, recent findings regarding the involvement of B cells and CD4+ T cells in hepatic immunity are also reviewed. Furthermore, we address the challenges ahead and propose integrating cutting-edge techniques, such as spatial biology, and combining mouse models with human sample analyses to gain comprehensive insights into the liver's adaptive immunity. This understanding could pave the way for novel therapeutic strategies targeting infectious diseases, malignancies, and inflammatory liver conditions like nonalcoholic steatohepatitis and autoimmune hepatitis. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Deterministic reprogramming of neutrophils within tumors.

Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1+ state. Reprogrammed dcTRAIL-R1+ neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies.

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.

Immunomodulatory leptin receptor+ sympathetic perineurial barrier cells protect against obesity by facilitating brown adipose tissue thermogenesis.

Adipose tissues (ATs) are innervated by sympathetic nerves, which drive reduction of fat mass via lipolysis and thermogenesis. Here, we report a population of immunomodulatory leptin receptor-positive (LepR+) sympathetic perineurial barrier cells (SPCs) present in mice and humans, which uniquely co-express Lepr and interleukin-33 (Il33) and ensheath AT sympathetic axon bundles. Brown ATs (BATs) of mice lacking IL-33 in SPCs (SPCΔIl33) had fewer regulatory T (Treg) cells and eosinophils, resulting in increased BAT inflammation. SPCΔIl33 mice were more susceptible to diet-induced obesity, independently of food intake. Furthermore, SPCΔIl33 mice had impaired adaptive thermogenesis and were unresponsive to leptin-induced rescue of metabolic adaptation. We therefore identify LepR+ SPCs as a source of IL-33, which orchestrate an anti-inflammatory BAT environment, preserving sympathetic-mediated thermogenesis and body weight homeostasis. LepR+IL-33+ SPCs provide a cellular link between leptin and immune regulation of body weight, unifying neuroendocrinology and immunometabolism as previously disconnected fields of obesity research.

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.

IL-1ß+ macrophages fuel pathogenic inflammation in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with high resistance to therapies1. Inflammatory and immunomodulatory signals co-exist in the pancreatic tumour microenvironment, leading to dysregulated repair and cytotoxic responses. Tumour-associated macrophages (TAMs) have key roles in PDAC2, but their diversity has prevented therapeutic exploitation. Here we combined single-cell and spatial genomics with functional experiments to unravel macrophage functions in pancreatic cancer. We uncovered an inflammatory loop between tumour cells and interleukin-1ß (IL-1ß)-expressing TAMs, a subset of macrophages elicited by a local synergy between prostaglandin E2 (PGE2) and tumour necrosis factor (TNF). Physical proximity with IL-1ß+ TAMs was associated with inflammatory reprogramming and acquisition of pathogenic properties by a subset of PDAC cells. This occurrence was an early event in pancreatic tumorigenesis and led to persistent transcriptional changes associated with disease progression and poor outcomes for patients. Blocking PGE2 or IL-1ß activity elicited TAM reprogramming and antagonized tumour cell-intrinsic and -extrinsic inflammation, leading to PDAC control in vivo. Targeting the PGE2-IL-1ß axis may enable preventive or therapeutic strategies for reprogramming of immune dynamics in pancreatic 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, 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.

The guanine nucleotide exchange factor Rin-like controls Tfh cell differentiation via CD28 signaling.

T follicular helper (Tfh) cells are essential for the development of germinal center B cells and high-affinity antibody-producing B cells in humans and mice. Here, we identify the guanine nucleotide exchange factor (GEF) Rin-like (Rinl) as a negative regulator of Tfh generation. Loss of Rinl leads to an increase of Tfh in aging, upon in vivo immunization and acute LCMV Armstrong infection in mice, and in human CD4+ T cell in vitro cultures. Mechanistically, adoptive transfer experiments using WT and Rinl-KO naïve CD4+ T cells unraveled T cell-intrinsic GEF-dependent functions of Rinl. Further, Rinl regulates CD28 internalization and signaling, thereby shaping CD4+ T cell activation and differentiation. Thus, our results identify the GEF Rinl as a negative regulator of global Tfh differentiation in an immunological context and species-independent manner, and furthermore, connect Rinl with CD28 internalization and signaling pathways in CD4+ T cells, demonstrating for the first time the importance of endocytic processes for Tfh differentiation.

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.

B cell class switch recombination is regulated by DYRK1A through MSH6 phosphorylation.

Protection from viral infections depends on immunoglobulin isotype switching, which endows antibodies with effector functions. Here, we find that the protein kinase DYRK1A is essential for B cell-mediated protection from viral infection and effective vaccination through regulation of class switch recombination (CSR). Dyrk1a-deficient B cells are impaired in CSR activity in vivo and in vitro. Phosphoproteomic screens and kinase-activity assays identify MSH6, a DNA mismatch repair protein, as a direct substrate for DYRK1A, and deletion of a single phosphorylation site impaired CSR. After CSR and germinal center (GC) seeding, DYRK1A is required for attenuation of B cell proliferation. These findings demonstrate DYRK1A-mediated biological mechanisms of B cell immune responses that may be used for therapeutic manipulation in antibody-mediated autoimmunity.

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.

Efficacy of mucosal vaccination using a protozoan parasite as a vehicle for antigen delivery: IgG and neutralizing response after rectal administration of LeCoVax-2, a candidate vaccine against COVID-19.

Mucosal vaccination is regarded as a promising alternative to classical, intramuscular vaccine delivery. However, only a limited number of vaccines have been licensed for mucosal administration in humans. Here we propose Leishmania tarentolae, a protozoan parasite, as a potential antigen vehicle for mucosal vaccination, for administration via the rectal or oral routes. To test this hypothesis, we exploited L. tarentolae for the production and delivery of SARS-CoV-2 antigens. Two antigens were assayed in BALB/c mice: Lt-spike, a L. tarentolae clone engineered for the surface expression of the SARS-CoV-2 spike protein; RBD-SD1, a purified portion of the spike protein, produced by another engineered clone of the protozoon. Immune response parameters were then determined at different time points. Both antigens, administered either separately or in combination (Lt-spike + RBD-SD1, hereafter LeCoVax-2), determined significant IgG seroconversion and production of neutralizing antibodies after subcutaneous administration, but only in the presence of adjuvants. After rectal administration, the purified RBD-SD1 antigen did not induce any detectable immune response, in comparison with the intense response observed after administration of LeCoVax-2 or Lt-spike alone. In rectal administration, LeCoVax-2 was also effective when administered without adjuvant. Our results show that L. tarentolae is an efficient and safe scaffold for production and delivery of viral antigens, to be used as vaccines. In addition, rectal vaccination experiments prove that L. tarentolae is suitable as a vaccine vehicle and adjuvant for enteral vaccination. Finally, the combined preparation LeCoVax-2 can be considered as a promising candidate vaccine against SARS-CoV-2, worthy of further investigation.

DNA-Vaccine-Induced Immune Response Correlates with Lower Viral SARS-CoV-2 Titers in a Ferret Model.

The COVID-19 pandemic is entering a new era with the approval of many SARS-CoV-2 vaccines. In spite of the restoration of an almost normal way of life thanks to the immune protection elicited by these innovative vaccines, we are still facing high viral circulation, with a significant number of deaths. To further explore alternative vaccination platforms, we developed COVID-eVax-a genetic vaccine based on plasmid DNA encoding the RBD domain of the SARS-CoV-2 spike protein. Here, we describe the correlation between immune responses and the evolution of viral infection in ferrets infected with the live virus. We demonstrate COVID-eVax immunogenicity as means of antibody response and, above all, a significant T-cell response, thus proving the critical role of T-cell immunity, in addition to the neutralizing antibody activity, in controlling viral spread.