Antiviral Effector Research Articles

MARCH8 Restricts RSV Replication by Promoting Cellular Apoptosis Through Ubiquitin-Mediated Proteolysis of Viral SH Protein

Numerous host factors function as intrinsic antiviral effectors to attenuate viral replication. MARCH8 is an E3 ubiquitin ligase that has been identified as a host restriction factor that inhibits the replication of various viruses. This study elucidated the mechanism by which MARCH8 restricts respiratory syncytial virus (RSV) replication through selective degradation of the viral small hydrophobic (SH) protein. We demonstrated that MARCH8 directly interacts with RSV-SH and catalyzes its ubiquitination at lysine 13, leading to SH degradation via the ubiquitin-lysosomal pathway. Functionally, MARCH8 expression enhances RSV-induced apoptosis through SH degradation, ultimately reducing viral titers. Conversely, an RSV strain harboring the SH-K13R mutation exhibited prolonged SH protein stability and attenuated apoptosis in infected cells, even in the presence of MARCH8. Targeted depletion of MARCH8 enhances cellular survival and potentially increases viral persistence. These findings demonstrate that MARCH8 promotes the early elimination of virus-infected cells by abrogating the anti-apoptotic function of SH, thereby reducing viral transmission. Our study provides novel insights into the interplay between host restriction factors and viral evasion strategies, potentially providing new therapeutic approaches for RSV infections.

An Auger electron-loaded theranostic biosensor triggered by the ACE2-mediated virus/host endocytosis

Accurate diagnosis and effective antiviral strategies are critical to combat acute infection and to avoid damage to the host. Due to their restricted radiation range and energy, Auger electron emitters have shown potential as a RNA-destructing radionuclide therapy in oncology and infection. Focusing on the process of angiotensin-converting enzyme 2 (ACE2)-mediated endocytosis, Technetium-99m-labeled DX600 (99mTc-DX600) was synthesized as an Auger electron vector to specifically bind to surface-expressed ACE2 proteins on 293T-hACE2 cells (293T cells stably expressing human ACE2), and Technetium-99m-loaded microvesicles (99mTc-MVs) served as an antiviral tracer and effector in pseudovirus infection. The whole-body ACE2 expression evaluation was non-invasive, meanwhile, the enhanced green fluorescent protein expression of pseudoviruses was substantially inhibited as a result of the 99mTc-DX600 loading of microvesicles, though the mitochondrial and DNA stabilities of the host cells were not affected. Furthermore, the in vivo distribution of 99mTc-DX600 in humanized ACE2 mice was demonstrated to be both ACE2-specific and long-lasting, and an antiviral effect was fully exhibited with two cycles of intravenous injection at a dosage of 37 MBq. Taking advantage of the ACE2-mediated interaction and natural trigger mechanism of virus-induced endocytosis, 99mTc-MV represents a theranostic biosensor of Auger electrons that can expose viral RNA to lethal amounts of radiation, with the host cells receiving no detrimental radiation.

Association of IL1RN VNTR and NKG2A polymorphisms with hepatitis E infection, a case study from western India.

Interleukin 1 receptor antagonist (IL1RN) is a competitive inhibitor of interleukin 1 (IL-1). Natural killer cells (NK cells) contribute to the elimination of viruses by their antiviral effector function, which depends on a balance between inhibitory and activating receptor genes such as NKG2D and NKG2A. Using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, the association of intronic single-nucleotide polymorphisms (SNPs) in these genes with viral infection were assessed in 111 patients with hepatitis E virus (HEV) infection and 222 HEV-naive healthy controls. An SNP in the IL1RN (VNTR) gene revealed allele 2 to be associated with protection against HEV infection (IL1RN *1/*1 vs. IL1RN *2/*2, OR = 0.26, 95% CI = 0.14-0.47, p < 0.001). Similarly, a polymorphism in the intronic region of NKG2A revealed an association with protection in a co-dominant model (A/A vs. A/G: OR = 0.40; 95% CI = 0.24-0.67; A/A vs. G/G: OR = 0.25; 95% CI = 0.10-0.57; p < 0.05) and an association with susceptibility in a dominant model (A/A + A/G vs. G/G: OR = 2.28; 95% CI = 1.06-4.93; p < 0.05) and a recessive model (AA vs. AG + GG: OR = 2.71; 95% CI = 1.66-4.48; p < 0.001). Our data suggest that genetic polymorphisms in host NKG2A and IL1RN have both protective and detrimental roles in HEV infection, although their impact on disease outcome remains unknown.

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Cell-mediated Ab Response Disrupted in MIS-C See article p. 1452 CHD7 Promotes Anti-viral Short-lived Effector CD8+ T Cells See article p. 1528

A novel HIV triple broadly neutralizing antibody (bNAb) combination-based passive immunization of infant rhesus macaques achieves durable protective plasma neutralization levels and mediates anti-viral effector functions.

To eliminate vertical HIV transmission and achieve therapy-free viral suppression among children living with HIV, novel strategies beyond antiretroviral therapy (ART) are necessary. Our group previously identified a triple broadly neutralizing antibody (bNAb) combination comprising of 3BNC117, PGDM1400 and PGT151 that mediates robust in vitro neutralization and non-neutralizing effector functions against a cross-clade panel of simian human immunodeficiency viruses (SHIVs). In this study, we evaluated the safety, pharmacokinetics, and antiviral potency of this bNAb combination in infant rhesus macaques (RMs). We demonstrate that subcutaneous infusion of the triple bNAb regimen was well tolerated in pediatric monkeys and resulted in durable systemic and mucosal distribution. Plasma obtained from passively-immunized RMs demonstrated potent HIV-neutralizing and Fc-mediated antiviral effector functions. Finally, using the predicted serum neutralization 80% inhibitory dilution titer (PT80) biomarker threshold of >200, which was recently identified as a surrogate endpoint for evaluation of the preventative efficacy of bNAbs against mucosal viral acquisition in human clinical trials, we demonstrated that our regimen has PT80>200 against a large panel of plasma and breast milk-derived HIV strains and cross-clade SHIV variants. This data will guide the development of combination bNAbs for eliminating vertical HIV transmission and for achieving ART-free viral suppression among children living with HIV.

Metabolic Dependency Shapes Bivalent Antiviral Response in Host Cells in Response to Poly:IC: The Role of Glutamine.

The establishment of effective antiviral responses within host cells is intricately related to their metabolic status, shedding light on immunometabolism. In this study, we investigated the hypothesis that cellular reliance on glutamine metabolism contributes to the development of a potent antiviral response. We evaluated the antiviral response in the presence or absence of L-glutamine in the culture medium, revealing a bivalent response hinging on cellular metabolism. While certain interferon-stimulated genes (ISGs) exhibited higher expression in an oxidative phosphorylation (OXPHOS)-dependent manner, others were surprisingly upregulated in a glycolytic-dependent manner. This metabolic dichotomy was influenced in part by variations in interferon-β (IFN-β) expression. We initially demonstrated that the presence of L-glutamine induced an enhancement of OXPHOS in A549 cells. Furthermore, in cells either stimulated by poly:IC or infected with dengue virus and Zika virus, a marked increase in ISGs expression was observed in a dose-dependent manner with L-glutamine supplementation. Interestingly, our findings unveiled a metabolic dependency in the expression of specific ISGs. In particular, genes such as ISG54, ISG12 and ISG15 exhibited heightened expression in cells cultured with L-glutamine, corresponding to higher OXPHOS rates and IFN-β signaling. Conversely, the expression of viperin and 2'-5'-oligoadenylate synthetase 1 was inversely related to L-glutamine concentration, suggesting a glycolysis-dependent regulation, confirmed by inhibition experiments. This study highlights the intricate interplay between cellular metabolism, especially glutaminergic and glycolytic, and the establishment of the canonical antiviral response characterized by the expression of antiviral effectors, potentially paving the way for novel strategies to modulate antiviral responses through metabolic interventions.

Administration of anti-HIV-1 broadly neutralizing monoclonal antibodies with increased affinity to Fcγ receptors during acute SHIVAD8-EO infection

Anti-HIV-1 broadly neutralizing antibodies (bNAbs) have the dual potential of mediating virus neutralization and antiviral effector functions through their Fab and Fc domains, respectively. So far, bNAbs with enhanced Fc effector functions in vitro have only been tested in NHPs during chronic simian-HIV (SHIV) infection. Here, we investigate the effects of administering in acute SHIVAD8-EO infection either wild-type (WT) bNAbs or bNAbs carrying the S239D/I332E/A330L (DEL) mutation, which increases binding to FcγRs. Emergence of virus in plasma and lymph nodes (LNs) was delayed by bNAb treatment and occurred earlier in monkeys given DEL bNAbs than in those given WT bNAbs, consistent with faster clearance of DEL bNAbs from plasma. DEL bNAb-treated monkeys had higher levels of circulating virus-specific IFNγ single-producing CD8+ CD69+ T cells than the other groups. In LNs, WT bNAbs were evenly distributed between follicular and extrafollicular areas, but DEL bNAbs predominated in the latter. At week 8 post-challenge, LN monocytes and NK cells from DEL bNAb-treated monkeys upregulated proinflammatory signaling pathways and LN T cells downregulated TNF signaling via NF-κB. Overall, bNAbs with increased affinity to FcγRs shape innate and adaptive cellular immunity, which may be important to consider in future strategies of passive bNAb therapy.

The pseudogene GBP1P1 suppresses influenza A virus replication by acting as a protein decoy for DHX9.

Recently, substantial evidence has demonstrated that pseudogene-derived long noncoding RNAs (lncRNAs) as regulatory RNAs have been implicated in basic physiological processes and disease development through multiple modes of functional interaction with DNA, RNA, and proteins. Here, we report an important role for GBP1P1, the pseudogene of guanylate-binding protein 1, in regulating influenza A virus (IAV) replication in A549 cells. GBP1P1 was dramatically upregulated after IAV infection, which is controlled by JAK/STAT signaling. Functionally, ectopic expression of GBP1P1 in A549 cells resulted in significant suppression of IAV replication. Conversely, silencing GBP1P1 facilitated IAV replication and virus production, suggesting that GBP1P1 is one of the interferon-inducible antiviral effectors. Mechanistically, GBP1P1 is localized in the cytoplasm and functions as a sponge to trap DHX9 (DExH-box helicase 9), which subsequently restricts IAV replication. Together, these studies demonstrate that GBP1P1 plays an important role in antagonizing IAV replication.IMPORTANCELong noncoding RNAs (lncRNAs) are extensively expressed in mammalian cells and play a crucial role as regulators in various biological processes. A growing body of evidence suggests that host-encoded lncRNAs are important regulators involved in host-virus interactions. Here, we define a novel function of GBP1P1 as a decoy to compete with viral mRNAs for DHX9 binding. We demonstrate that GBP1P1 induction by IAV is mediated by JAK/STAT activation. In addition, GBP1P1 has the ability to inhibit IAV replication. Importantly, we reveal that GBP1P1 acts as a decoy to bind and titrate DHX9 away from viral mRNAs, thereby attenuating virus production. This study provides new insight into the role of a previously uncharacterized GBP1P1, a pseudogene-derived lncRNA, in the host antiviral process and a further understanding of the complex GBP network.

Expression of tumor antigens within an oncolytic virus enhances the anti-tumor T cell response

Although patients benefit from immune checkpoint inhibition (ICI) therapy in a broad variety of tumors, resistance may arise from immune suppressive tumor microenvironments (TME), which is particularly true of hepatocellular carcinoma (HCC). Since oncolytic viruses (OV) can generate a highly immune-infiltrated, inflammatory TME, OVs could potentially restore ICI responsiveness via recruitment, priming, and activation of anti-tumor T cells. Here we find that on the contrary, an oncolytic vesicular stomatitis virus, expressing interferon-ß (VSV-IFNß), antagonizes the effect of anti-PD-L1 therapy in a partially anti-PD-L1-responsive model of HCC. Cytometry by Time of Flight shows that VSV-IFNß expands dominant anti-viral effector CD8 T cells with concomitant relative disappearance of anti-tumor T cell populations, which are the target of anti-PD-L1. However, by expressing a range of HCC tumor antigens within VSV, combination OV and anti-PD-L1 therapeutic benefit could be restored. Our data provide a cautionary message for the use of highly immunogenic viruses as tumor-specific immune-therapeutics by showing that dominant anti-viral T cell responses can inhibit sub-dominant anti-tumor T cell responses. However, through encoding tumor antigens within the virus, oncolytic virotherapy can generate anti-tumor T cell populations upon which immune checkpoint blockade can effectively work.

Therapeutic potential of co-signaling receptor modulation in hepatitis B

Reversing CD8+ Tcell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ Tcells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ Tcells. Targeting all dysfunctional Tcells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.

The balance between antiviral and antibacterial responses during M. tuberculosis infection is regulated by the ubiquitin ligase CBL.

As a first line of host defense, macrophages must be able to effectively sense and respond to diverse types of pathogens, and while a particular type of immune response may be beneficial in some circumstances, it can be detrimental in others. Upon infecting a macrophage, M. tuberculosis (Mtb) induces proinflammatory cytokines that activate antibacterial responses. Surprisingly, Mtb also triggers antiviral responses that actually hinder the ability of macrophages to control Mtb infection. The ubiquitin ligase CBL suppresses these antiviral responses and shifts macrophages toward a more antibacterial state during Mtb infection, however, the mechanisms by which CBL regulates immune signaling are unknown. We found that CBL controls responses to multiple stimuli and broadly suppresses the expression of antiviral effector genes. We then used mass-spectrometry to investigate potential CBL substrates and identified over 46,000 ubiquitylated peptides in Mtb-infected macrophages, as well as roughly 400 peptides with CBL-dependent ubiquitylation. We then performed genetic interaction analysis of CBL and its putative substrates, and identified the Fas associated factor 2 (FAF2) adapter protein as a key signaling molecule protein downstream of CBL. Together, these analyses identify thousands of new ubiquitin-mediated signaling events during the innate immune response and reveal an important new regulatory hub in this response.

The immune response to RNA suppresses nucleic acid synthesis by limiting ribose 5-phosphate

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.

A non-canonical mechanism of antiviral activity of Oligoadenylate Synthetase 1

Abstract The translational arrest of protein synthesis is often a cellular response to the virus infection. However, some antiviral proteins continue to be translated through unknown mechanisms during this translational shutdown. Here, we report a mechanism by which antiviral effectors are upregulated during this translational shutdown. We found that interferon (IFN) stimulated gene, Oligoadenylate Synthetase 1 (OAS1), binds AU-rich elements (ARE) of specific mRNA, including IFNβ, prolonging the half-life and continued expression. This increased IFN expression protects from WNV infection in vitro and in vivo via downstream IFNAR signaling. This mechanism is common between human OAS1 and mouse Oas1b, independent of OAS enzyme activity and RNase L. However, human OAS1 inhibits SARS-CoV-2 replication through its canonical enzyme activity via RNase L, thus establishing two different mechanisms of OAS1 antiviral activity. These results establish OAS1 as an ARE-binding protein with a broader non-canonical function that protects IFN expression from translational shutdown.

Targeting the NS4b Antigen of West Nile Virus to the Endocytic Receptor DEC-205 to Stimulate Protective T Cell Responses to West Nile Virus Encephalitis

Abstract West Nile Virus (WNV), the primary cause of mosquito-borne neuroinvasive disease in the US, poses a significant public health threat without an approved human vaccine. DCs are pivotal in orchestrating immune responses, dictating antigen-specific effector T cell activation or tolerance based on their maturity. The endocytic receptor DEC-205, expressed by specific DC subsets, facilitates antigen uptake, processing, and cross-presentation, initiating an effective T cell response. Studies highlight DEC-205+ DCs within the CNS in reactivating antiviral effector T cell responses, restricting WNV replication, and mitigating immunopathology. We propose targeting antigens to these DCs to enhance antiviral immunity against WNV within the CNS, fusing antigens to DEC-205-specific monoclonal antibodies (mAbs). A DEC-205-targeted hybrid antibody will be developed, conjugating the WNV NS4b immunodominant peptide to a DEC-205 mAb. Subsequent purification will precede testing therapeutic efficacy in a murine WNV encephalitis model. Delivering the aDEC-205 hybrid mAb with DC maturation stimulus (poly I:C), we will assess co-stimulatory molecule expression (CD40, CD80, and CD86) on DCs and evaluate T cell activation through activation marker expression and cytokine production. This research introduces a vaccination approach against West Nile Virus encephalitis, leveraging targeted antigen delivery to DEC-205+ DCs, offering insights into potential therapeutic and preventive strategies.

Therapeutic prime/pull vaccination of HSV-2-infected guinea pigs with the ribonucleotide reductase 2 (RR2) protein and CXCL11 chemokine boosts antiviral local tissue-resident and effector memory CD4+ and CD8+ T cells and protects against recurrent genital herpes.

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and its effect on the frequency and severity of recurrent genital herpes in the recurrent herpes guinea pig model. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-2 ribonucleotide reductase 2 (RR2) protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 chemokines to recruit CD4+ and CD8+ T cells into the infected DRG and VM (Pull). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident and effector memory CD4+ and CD8+ T cells in both DRG and VM tissues. This was associated with less virus in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4+ and CD8+ T cells in reducing virus shedding at the vaginal site of infection and the severity of recurrent genital herpes and propose the novel prime-pull vaccine strategy to protect against recurrent genital herpes.IMPORTANCEThe present study investigates the novel prime/pull therapeutic vaccine strategy to protect against recurrent genital herpes using the latently infected guinea pig model. In this study, we used the strategy that involves immunization of herpes simplex virus type 2-infected guinea pigs using a recombinantly expressed herpes tegument protein-ribonucleotide reductase 2 (RR2; prime), followed by intravaginal treatment with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines to recruit T cells into the infected dorsal root ganglia (DRG) and vaginal mucosa (VM) (pull). We show that the RR2/CXCL11 prime-pull therapeutic vaccine strategy elicited a significant reduction in virus shedding in the vaginal mucosa and decreased the severity and frequency of recurrent genital herpes. This protection was associated with increased frequencies of functional tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells infiltrating latently infected DRG tissues and the healed regions of the vaginal mucosa. These findings shed light on the role of tissue-resident and effector memory CD4+ and CD8+ T cells in DRG tissues and the VM in protection against recurrent genital herpes and propose the prime-pull therapeutic vaccine strategy in combating genital herpes.

Investigating the Evolution of Drosophila STING-Dependent Antiviral Innate Immunity by Multispecies Comparison of 2'3'-cGAMP Responses.

Viruses represent a major threat to all animals, which defend themselves through induction of a large set of virus-stimulated genes that collectively control the infection. In vertebrates, these genes include interferons that play a critical role in the amplification of the response to infection. Virus- and interferon-stimulated genes include restriction factors targeting the different steps of the viral replication cycle, in addition to molecules associated with inflammation and adaptive immunity. Predictably, antiviral genes evolve dynamically in response to viral pressure. As a result, each animal has a unique arsenal of antiviral genes. Here, we exploit the capacity to experimentally activate the evolutionarily conserved stimulator of IFN genes (STING) signaling pathway by injection of the cyclic dinucleotide 2'3'-cyclic guanosine monophosphate-adenosine monophosphate into flies to define the repertoire of STING-regulated genes in 10 Drosophila species, spanning 40 million years of evolution. Our data reveal a set of conserved STING-regulated factors, including STING itself, a cGAS-like-receptor, the restriction factor pastel, and the antiviral protein Vago, but also 2 key components of the antiviral RNA interference pathway, Dicer-2, and Argonaute2. In addition, we identify unknown species- or lineage-specific genes that have not been previously associated with resistance to viruses. Our data provide insight into the core antiviral response in Drosophila flies and pave the way for the characterization of previously unknown antiviral effectors.

Modulation of cytomegalovirus immune evasion identifies direct antigen presentation as the predominant mode of CD8 T-cell priming during immune reconstitution after hematopoietic cell transplantation.

Cytomegalovirus (CMV) infection is the most critical infectious complication in recipients of hematopoietic cell transplantation (HCT) in the period between a therapeutic hematoablative treatment and the hematopoietic reconstitution of the immune system. Clinical investigation as well as the mouse model of experimental HCT have consistently shown that timely reconstitution of antiviral CD8 T cells is critical for preventing CMV disease in HCT recipients. Reconstitution of cells of the T-cell lineage generates naïve CD8 T cells with random specificities among which CMV-specific cells need to be primed by presentation of viral antigen for antigen-specific clonal expansion and generation of protective antiviral effector CD8 T cells. For CD8 T-cell priming two pathways are discussed: "direct antigen presentation" by infected professional antigen-presenting cells (pAPCs) and "antigen cross-presentation" by uninfected pAPCs that take up antigenic material derived from infected tissue cells. Current view in CMV immunology favors the cross-priming hypothesis with the argument that viral immune evasion proteins, known to interfere with the MHC class-I pathway of direct antigen presentation by infected cells, would inhibit the CD8 T-cell response. While the mode of antigen presentation in the mouse model of CMV infection has been studied in the immunocompetent host under genetic or experimental conditions excluding either pathway of antigen presentation, we are not aware of any study addressing the medically relevant question of how newly generated naïve CD8 T cells become primed in the phase of lympho-hematopoietic reconstitution after HCT. Here we used the well-established mouse model of experimental HCT and infection with murine CMV (mCMV) and pursued the recently described approach of up- or down-modulating direct antigen presentation by using recombinant viruses lacking or overexpressing the central immune evasion protein m152 of mCMV, respectively. Our data reveal that the magnitude of the CD8 T-cell response directly reflects the level of direct antigen presentation.

Evolutionary and functional conservation of IRF7 in the Tibetan frog Nanorana parkeri.

The production of interferons (IFNs) is essential for the control of viral infections, and interferon regulatory factor 7 (IRF7) is considered as a vital regulator for the transcription of type I IFNs. Amphibians appear to possess a highly expanded type I IFN repertoire, consisting of intron-containing genes as observed in fish, and intronless genes as in other higher vertebrates. However, the knowledge on transcriptional regulatory mechanism of these two types of type I IFN genes is rather scarce in amphibians. A IRF7 gene named as Np-IRF7 was identified in Tibetan frog (Nanorana parkeri), and bioinformatic analysis revealed that the predicted protein of Np-IRF7 contains several important structural features known in IRF7. Expression analysis showed that Np-IRF7 gene was widely expressed and rapidly induced by poly(I:C) in different organs/tissues. Interestingly, luciferase reporter assay revealed that intronless IFN promoters were more effectively activated than intron-containing IFN promoter in Np-IRF7-transfected cells. Moreover, the overexpression of Np-IRF7 could induce the expression of ISGs and suppress the replication of FV3 in A6 cells. Np-IRF7 is indeed the ortholog of known IRF7, and IRF7 is structurally conserved in different lineages of vertebrates. Np-IRF7 played distinct roles in the activation of intron-containing and intronless type I IFN promoters, thus inducing the expression of interferon-stimulated antiviral effectors and providing a protection against ranavirus infection. The present research thus contributes to a better understanding of regulatory function of IRF7 in the IFN-mediated antiviral response of anuran amphibians.

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.

A type 1 immunity-restricted promoter of the IL−33 receptor gene directs antiviral T-cell responses

The pleiotropic alarmin interleukin-33 (IL-33) drives type 1, type 2 and regulatory T-cell responses via its receptor ST2. Subset-specific differences in ST2 expression intensity and dynamics suggest that transcriptional regulation is key in orchestrating the context-dependent activity of IL-33–ST2 signaling in T-cell immunity. Here, we identify a previously unrecognized alternative promoter in mice and humans that is located far upstream of the curated ST2-coding gene and drives ST2 expression in type 1 immunity. Mice lacking this promoter exhibit a selective loss of ST2 expression in type 1- but not type 2-biased T cells, resulting in impaired expansion of cytotoxic T cells (CTLs) and T-helper 1 cells upon viral infection. T-cell-intrinsic IL-33 signaling via type 1 promoter-driven ST2 is critical to generate a clonally diverse population of antiviral short-lived effector CTLs. Thus, lineage-specific alternative promoter usage directs alarmin responsiveness in T-cell subsets and offers opportunities for immune cell-specific targeting of the IL-33–ST2 axis in infections and inflammatory diseases.