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Abstract Background Liver transplant recipients (LTRs) are at a high risk of severe COVID-19 owing to immunosuppression and comorbidities. LTRs are less responsive to mRNA vaccines than healthy donors (HDs) or other immunosuppressed patients. However, the disruption mechanism in humoral and cellular immune memory responses is unclear. Methods We longitudinally collected peripheral blood mononuclear cells and plasma samples from HDs (n = 44) and LTRs (n = 54) who received BNT162b2 or mRNA-1273 vaccines. We measured the levels of anti-receptor-binding domain (RBD) antibodies and spike-specific CD4+ and CD8+ T-cell responses. Results Here, we show that the induction of anti-RBD IgG was weaker in LTRs than in HDs. The use of multiple immunosuppressive drugs is associated with lower antibody titers than only calcineurin inhibitor, and limits the induction of CD4+ T-cell responses. However, spike-specific CD4+ T-cell and antibody responses improved with a third vaccination. Furthermore, mRNA vaccine-induced spike-specific CD8+ T cells are quantitatively, but not qualitatively, limited to LTRs. Both CD4+ and CD8+ T cells react to omicron sublineages, regardless of the presence in HDs or LTRs. However, there is no boosting effect of spike-specific memory CD8+ T-cell responses after a third vaccination in HDs or LTRs. Conclusions The third mRNA vaccination improves both humoral responses and spike-specific CD4+ T-cell responses in LTRs but provides no booster effect for spike-specific memory CD8+ T-cell responses. A third mRNA vaccination could be helpful in LTRs to prevent severe COVID-19, although further investigation is required to elicit CD8+ T-cell responses in LTRs and HDs.

CD8+ T cells affect the outcomes of pancreatic ductal adenocarcinoma (PDAC). Using tissue samples at pre-treatment to monitor the immune response is challenging, while blood samples are beneficial in overcoming this limitation. In this study, we measured peripheral antigen-specific CD8+ T cell responses against four different tumor-associated antigens (TAAs) in PDAC using flow cytometry and investigated their relationships with clinical features. We analyzed the optimal timing within the treatment course for effective immune checkpoint inhibition in vitro. We demonstrated that the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells was correlated with a fold reduction in CA19-9 before and after neoadjuvant therapy. Moreover, patients with TAA-specific IFNγ+4-1BB+ CD8+ T cells after surgery exhibited a significantly improved disease-free survival. Anti-PD-1 treatment in vitro increased the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells before neoadjuvant therapy in patients, suggesting the importance of the timing of anti-PD-1 inhibition during the treatment regimen. Our results indicate that peripheral immunophenotyping, combined with highly sensitive identification of TAA-specific responses in vitro as well as detailed CD8+ T cell subset profiling via ex vivo analysis, may serve as peripheral biomarkers to predict treatment outcomes and therapeutic efficacy of immunotherapy plus neoadjuvant chemotherapy.

Stimulator of interferon genes (STING) is a cytoplasmic dinucleotide sensor used as an immunomodulatory agent for cancer treatment. The efficacy of the STING ligand (STING-L) against various tumors has been evaluated in mouse models; however, its safety and efficacy in non-human primates have not been reported. We examined the effects of escalating doses of cyclic-di-adenosine monophosphate (c-di-AMP) or cyclic [G (3′,5′)pA (3′,5′p] (3′-3′-cGAMP) administered intramuscularly or intravenously to cynomolgus macaques. Both ligands induced transient local and systemic inflammatory responses and systemic immunomodulatory responses, including the upregulation of interferon-α (IFN-α) and IFN-γ expression and the activation of multiple immunocompetent cell subsets. Better immunological responses were observed in animals that received c-di-AMP compared with those that received 3′-3′-cGAMP. Multi-parameter analysis using a dataset obtained before administering the ligands predicted the efficacy outcome partially. Importantly, the efficacy of these ligands was reduced in older macaques. We propose that 0.5 mg/kg c-di-AMP via intramuscular administration should be the optimal starting point for clinical studies. Our study is the first to demonstrate the age-dependent safety and efficacy of STING-L in non-human primates and supports the potential of STING-L use as a direct immunomodulator in vivo.

Despite treatment, hepatitis B surface antigen (HBsAg) persists in patients with chronic hepatitis B (CHB), suggesting the likely presence of the virus in the body. CD8+ T cell responses are essential for managing viral replication, but their effect on HBsAg levels remains unclear. We studied the traits of activated CD8+ T cells and HBV-specific CD8+ T cells in the blood of CHB patients undergoing nucleos(t)ide analog (NUC) therapy. For the transcriptome profiling of activated CD8+ T cells in peripheral blood mononuclear cells (PBMCs), CD69+ CD8+ T cells were sorted from six donors, and single-cell RNA sequencing (scRNA-seq) analysis was performed. To detect HBV-specific CD8+ T cells, we stimulated PBMCs from 26 donors with overlapping peptides covering the HBs, HBcore, and HBpol regions of genotype A/B/C viruses, cultured for 10 days, and analyzed via multicolor flow cytometry. scRNA-seq data revealed that CD8+ T cell clusters harboring the transcripts involved in the cytolytic functions were frequently observed in donors with high HBsAg levels. Polyfunctional analysis of HBV-specific CD8+ T cells utilized by IFN-γ/TNFα/CD107A/CD137 revealed that HBcore-specific cells exhibited greater polyfunctionality, suggesting that the quality of HBV-specific CD8+ T cells varies among antigens. Moreover, a subset of HBcore-specific CD8+ T cells with lower cytolytic potential was inversely correlated with HBsAg level. Our results revealed a stimulant-dependent qualitative difference in HBV-specific CD8+ T cells in patients with CHB undergoing NUC therapy. Hence, the induction of HBcore-specific CD8+ T cells with lower cytolytic potential could be a new target for reducing HBsAg levels.

Abstract The quality and size of liver grafts are critical factors that influence living‐donor liver transplantation (LDLT) function and safety. However, the biomarkers used for predicting graft quality are lacking. In this study, we sought to identify unique graft quality markers, aside from donor age, by using the livers of non‐human primates. Hepatic gene microarray expression data from young and elderly cynomolgus macaques revealed a total of 271 genes with significantly increased expression in the elderly. These candidate genes were then narrowed down to six through bioinformatics analyses. The expression patterns of these candidate genes in human donor liver tissues were subsequently examined. Importantly, we found that grafts exhibiting up‐regulated expression of these six candidate genes were associated with an increased incidence of liver graft failure. Multivariable analysis further revealed that up‐regulated expression of LRRN2 (encoding leucine‐rich repeat protein, neuronal 2) in donor liver tissue served as an independent risk factor for graft failure (odds ratio 4.50, confidence interval 2.08–9.72). Stratification based on graft expression of LRRN2 and donor age was also significantly associated with 6‐month graft survival rates. Conclusion: Up‐regulated LRRN2 expression of liver graft is significantly correlated with graft failure in LDLT. In addition, combination of graft LRRN2 expression and donor age may represent a promising marker for predicting LDLT graft quality.

Understanding the T-cell responses involved in inhibiting COVID-19 severity is crucial for developing new therapeutic and vaccine strategies. Here, we characterized SARS-CoV-2 spike-specific CD8+ T cells in vaccinees longitudinally. The BNT162b2 mRNA vaccine can induce spike-specific CD8+ T cells cross-reacting to BA.1, whereas the T-cell receptor (TCR) repertoire usages decreased with time. Furthermore the mRNA vaccine induced spike-specific CD8+ T cells subpopulation expressing Granzyme A (GZMA), Granzyme B (GZMB) and Perforin simultaneously in healthy donors at 4 weeks after the second vaccination. The induced subpopulation was not maintained at 12 weeks after the second vaccination. Incorporating factors that efficiently induce CD8+ T cells with highly cytotoxic activity could improve future vaccine efficacy against such variants.

Summary: Efficient delivery of subunit vaccines to dendritic cells (DCs) is necessary to improve vaccine efficacy, because the vaccine antigen alone cannot induce sufficient protective immunity. Here, we identified DC-targeting peptides using a phage display system and demonstrated the potential of these peptides as antigen-delivery carriers to improve subunit vaccine effectiveness in mice. The fusion of antigen proteins and peptides with DC-targeting peptides induced strong antigen-specific IgG responses, even in the absence of adjuvants. In addition, the DC-targeting peptide improved the distribution of antigens to DCs and antigen presentation by DCs. The combined use of an adjuvant with a DC-targeting peptide improved the effectiveness of the vaccine. Furthermore, nucleolin, located on the DC surface, was identified as the receptor for DC-targeting peptide, and nucleolin was indispensable for the vaccine effect of the DC-targeting peptide. Overall, the findings of this study could be useful for developing subunit vaccines against infectious diseases.

Summary: Several cross-protective antibodies that recognize a broad range of influenza A virus (IAV) strains are known to have functions in virus elimination such as Fcγ receptor (FcγR)-effector function and neutralizing activity against the head region. Although few studies have used primary cells as effector cells, the FcγR-effector function was evaluated after isolating each cell subset. Herein, we established an original assay system to evaluate purified FI6 IgG-mediated binding to hemagglutinin (HA)-expressing cells by flow cytometry using peripheral blood mononuclear cells from cynomolgus macaques. In addition, we evaluated the FcγR-effector function of IAV vaccine-induced anti-HA antibodies in cynomolgus macaques after administering the split vaccine. We found several cell types, mainly classical monocytes, bound to HA-expressing target cells in an FcγR-dependent manner, that were dominant in the binding of the cell population. Thus, this assay system could facilitate the development of a universal influenza vaccine.

Abstract Background Recent data suggest the importance of non-neutralizing antibodies (nnAbs) in the development of vaccines against HIV-1 because two types of nnAbs that recognize the coreceptor binding site (CoRBS) and the C1C2 region mediate antibody-dependent cellular-cytotoxicity (ADCC) against HIV-1-infected cells. However, many studies have been conducted with nnAbs obtained from subtype B-infected individuals, with few studies in patients with non-subtype B infections. Results We isolated a monoclonal antibody 1E5 from a CRF02_AG-infected individual and constructed two forms of antibody with constant regions of IgG1 or IgG3. The epitope of 1E5 belongs to the C1C2 of gp120, and 1E5 binds to 27 out of 35 strains (77 %) across the subtypes. The 1E5 showed strong ADCC activity, especially in the form of IgG3 in the presence of small CD4-mimetic compounds (CD4mc) and 4E9C (anti-CoRBS antibody), but did not show any neutralizing activity even against the isolates with strong binding activities. The enhancement in the binding of A32, anti-C1C2 antibody isolated from a patient with subtype B infection, was observed in the presence of 1E5 and the combination of 1E5, A32 and 4E9C mediated a strong ADCC activity. Conclusions These results suggest that anti-C1C2 antibodies that are induced in patients with different HIV-1 subtype infections have common functional modality and may have unexpected interactions. These data may have implications for vaccine development against HIV-1. Graphical abstract

Toll-like receptors (TLRs) were first identified as molecular sensors that transduce signals from specific structural patterns derived from pathogens; their underlying molecular mechanisms of recognition and signal transduction are well-understood. To date, more than 20 pattern-recognition receptors (PRRs) have been reported in humans, some of which are membrane-bound, similar to TLRs, whereas others are cytosolic, including retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and stimulator of interferon genes (STING). Clinically, PRR ligands have been developed as vaccine adjuvants to activate innate immunity and enhance subsequent antigen-specific immune responses. Recently, PRR ligands have been used as direct immunostimulators to enhance immune responses against infectious diseases and cancers. HIV-1 remains one of the world's most significant public health challenges. Without the elimination of HIV-1 latently infected cells, patients require lifelong combination antiretroviral therapy (cART), while research aimed at a functional cure for HIV-1 infection continues. Based on the concept of “shock and kill,” a latency-reversing agent (LRA) has been developed to reactivate latently infected cells and induce cell death. However, previous research has shown that LRAs have limited efficacy in the eradication of these reservoirs in vivo. Besides, PRR ligands with anti-retroviral drugs have been developed for use in HIV treatment for these years. This mini-review summarizes the current understanding of the role of PRR ligands in AIDS research, suggests directions for future research, and proposes potential clinical applications.

Abstract Background The CD4-induced (CD4i) epitopes in gp120 includes the co-receptor binding site, which are formed and exposed after interaction with CD4. Monoclonal antibodies (mAbs) to the CD4i epitopes exhibit limited neutralizing activity because of restricted access to their epitopes. However, small fragment counterparts such as single-chain variable fragments (scFvs) have been reported to neutralize a broad range of viruses compared with the full-size IgG molecule. To identify the CD4i epitope site responsible for this broad neutralization we constructed three scFvs of anti-CD4i mAbs from a human immunodeficiency virus type 1 (HIV-1)-infected elite controller, and investigated the neutralization coverage and precise binding site in the CD4i epitopes. Results We constructed scFvs from the anti-CD4i mAbs, 916B2, 4E9C, and 25C4b and tested their neutralization activity against a panel of 66 viruses of multi-subtype. Coverage of neutralization by the scFvs against this panel of pseudoviruses was 89% (59/66) for 4E9C, 95% (63/66) for 25C4b and 100% (66/66) for 916B2. Analysis using a series of envelope glycoprotein mutants revealed that individual anti-CD4i mAbs showed various dependencies on the hairpin 1 (H1) and V3 base. The binding profiles of 25C4b were similar to those of 17b, and 25C4b bound the region spanning multiple domains of H1 and hairpin 2 (H2) of the bridging sheet and V3 base. For 4E9C, the V3-base dependent binding was apparent from no binding to mutants containing the ΔV3 truncation. In contrast, binding of 916B2 was dependent on the H1 region, which is composed of β2 and β3 strands, because mutants containing the H1 truncation did not show any reactivity to 916B2. Although the H1 region structure is affected by CD4 engagement, the results indicate the unique nature of the 916B2 epitope, which may be structurally conserved before and after conformational changes of gp120. Conclusions Identification of a unique structure of the H1 region that can be targeted by 916B2 may have an important implication in the development of small molecules to inhibit infection by a broad range of HIV-1 for the purpose of HIV treatment and prevention.

Cell-to-cell spread of HIV permits ongoing viral replication in the presence of antiretroviral therapy and is suggested to be a major contributor to sexual transmission by mucosal routes. Fusion inhibitors that prevent viral entry have been developed, but their clinical applications have been limited by weak antiviral activity, short half-life, and the low genetic barrier to development of resistance. We examined the inhibitory activities of a series of single-chain variable fragments (scFvs) targeting the V3 and CD4i epitopes against both cell-free and cell-to-cell HIV infection. We found that all anti-V3 scFvs, including two newly constructed scFvs, showed broad neutralization activity against a panel of subtype B viruses compared with the corresponding IgGs. All scFvs neutralized cell-free infection by HIV-1JR-FL WT and fusion inhibitor-resistant mutants. In addition, all anti-V3 scFvs and some CD4i scFvs significantly inhibited cell fusion, while their IgG counterparts did not. Furthermore, scFvs-fusion inhibitors combinations, such as C34 and SC34, showed synergistic inhibition of cell fusion by both HIV-1JR-FL WT and fusion inhibitor-resistant mutants. The most prominent combinational effect was observed for 916B2 CD4i scFv with SC34. The delayed fusion kinetics of fusion inhibitor-resistant mutants partly explain their synergistic inhibition by such combinations. Our data demonstrate the advantages of using scFvs over their parent IgGs for inhibiting both cell-free and cell-to-cell infection. High synergistic inhibition of cell fusion by using scFvs-fusion inhibitors combinations suggests the possibility of intensification therapy adding this combination to current anti-HIV treatment regimens. Keywords: HIV-1, scFv, Fusion inhibitor, Neutralization, Cell-to cell infection

The attack and defense of infected cells and cytotoxic CD8 T cells occur in germinal centers in lymphoid tissue in chronic persistent HIV/SIV infection. Latently infected cells, the therapeutic target of HIV infection, accumulate in follicular helper T (Tfh) cells in lymphoid tissue; the impact of HIV-specific follicular CD8 (fCD8) T cells in lymphoid tissue on the latently infected cells remains unknown. We infected 15 cynomolgus macaques with SIVmac239 and examined the contribution of SIV-Gag-specific fCD8 T cells, defined by activation-induced markers (AIMs), to SIV-infected cells. Eight out of the 15 infected macaques served as progressors; a chronic phase combination antiretroviral therapy (cART) model was established for the eight macaques (progressors) with chronic persistent infection status, wherein cART was started in the chronic phase and discontinued after 27 weeks. Seven macaques that naturally controlled the viremia served as natural controllers. The frequency of SIV-G ag-specific fCD8 T cells was inversely correlated with the amount of cell-associated SIV-gag RNA in the Tfh only under cART or in the controllers but not in untreated progressors. scRNA-seq of SIV-Gag-specific fCD8 T cells in various conditions revealed that the gene expression pattern of SIV-Gag-specific fCD8 T cells in the controllers was closer to that of those under cART than the untreated progressors. Comparing the SIV-Ga g-specific fCD8 T cells of those under cART to the controllers revealed their more exhausted and immunosenescent nature under cART. Improving the HIV/SIV-specific fCD8 T cells under cART by targeting those pathways might contribute to the development of potential curative strategies. IMPORTANCE We infected cynomolgus macaques with SIVmac239 to establish an SIV-chronically infected cART model. We performed an in-depth characterization of Tfh and fCD8 T cells in three conditions--chronic stage of untreated, cART-treated, and natural controller cynomolgus macaques--by combining tissue section analysis and single-cell analyses of sorted cells. We revealed the inverse relationship between Tfh infection and SIV-Gag-specific fCD8 T cell frequencies as observed in HIV-infected individuals, thereby establishing the cynomolgus macaque as a relevant animal model to study the determinants of HIV/SIV persistence in lymphoid tissue. Additionally, scRNAseq analysis of SIV-Gag-specific fCD8 T cells revealed an enrichment of exhausted or senescent transcriptomic signatures under cART. These data will provide the basic insights into virus-host CD8 T cell interactions, particularly within the follicular region, during latent HIV infection under ART. [ABSTRACT FROM AUTHOR]

CD8+ T cells affect the outcomes of pancreatic ductal adenocarcinoma (PDAC). Using tissue samples at pre-treatment to monitor the immune response is challenging, while blood samples are beneficial in overcoming this limitation. In this study, we measured peripheral antigen-specific CD8+ T cell responses against four different tumor-associated antigens (TAAs) in PDAC using flow cytometry and investigated their relationships with clinical features. We analyzed the optimal timing within the treatment course for effective immune checkpoint inhibition in vitro. We demonstrated that the frequency of TAAspecific IFNγ+4-1BB+ CD8+ T cells was correlated with a fold reduction in CA19-9 before and after neoadjuvant therapy. Moreover, patients with TAA-specific IFNγ+4-1BB+ CD8+ T cells after surgery exhibited a significantly improved disease-free survival. Anti-PD-1 treatment in vitro increased the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells before neoadjuvant therapy in patients, suggesting the importance of the timing of anti-PD-1 inhibition during the treatment regimen. Our results indicate that peripheral immunophenotyping, combined with highly sensitive identification of TAA-specific responses in vitro as well as detailed CD8+ T cell subset profiling via ex vivo analysis, may serve as peripheral biomarkers to predict treatment outcomes and therapeutic efficacy of immunotherapy plus neoadjuvant chemotherapy. [ABSTRACT FROM AUTHOR]

Despite treatment, hepatitis B surface antigen (HBsAg) persists in patients with chronic hepatitis B (CHB), suggesting the likely presence of the virus in the body. CD8+ T cell responses are essential for managing viral replication, but their effect on HBsAg levels remains unclear. We studied the traits of activated CD8+ T cells and HBV-specific CD8+ T cells in the blood of CHB patients undergoing nucleos (t)ide analog (NUC) therapy. For the transcriptome profiling of activated CD8+ T cells in peripheral blood mononuclear cells (PBMCs), CD69+ CD8+ T cells were sorted from six donors, and single-cell RNA sequencing (scRNA-seq) analysis was performed. To detect HBV-specific CD8+ T cells, we stimulated PBMCs from 26 donors with overlapping peptides covering the HBs, HBcore, and HBpol regions of genotype A/B/C viruses, cultured for 10 days, and analyzed via multicolor flow cytometry. scRNA-seq data revealed that CD8+ T cell clusters harboring the transcripts involved in the cytolytic functions were frequently observed in donors with high HBsAg levels. Polyfunctional analysis of HBVspecific CD8+ T cells utilized by IFN-γ/TNFα/CD107A/CD137 revealed that HBcore-specific cells exhibited greater polyfunctionality, suggesting that the quality of HBV-specific CD8+ T cells varies among antigens. Moreover, a subset of HBcore-specific CD8+ T cells with lower cytolytic potential was inversely correlated with HBsAg level. Our results revealed a stimulant-dependent qualitative difference in HBV-specific CD8+ T cells in patients with CHB undergoing NUC therapy. Hence, the induction of HBcore-specific CD8+ T cells with lower cytolytic potential could be a new target for reducing HBsAg levels. [ABSTRACT FROM AUTHOR]

Understanding the T-cell responses involved in inhibiting COVID-19 severity is crucial for developing new therapeutic and vaccine strategies. Here, we characterized SARS-CoV-2 spike-specific CD8+ T cells in vaccinees longitudinally. The BNT162b2 mRNA vaccine can induce spike-specific CD8+ T cells cross-reacting to BA.1, whereas the T-cell receptor (TCR) repertoire usages decreased with time. Furthermore the mRNA vaccine induced spikespecific CD8+ T cells subpopulation expressing Granzyme A (GZMA), Granzyme B (GZMB) and Perforin simultaneously in healthy donors at 4 weeks after the second vaccination. The induced subpopulation was not maintained at 12 weeks after the second vaccination. Incorporating factors that efficiently induce CD8+ T cells with highly cytotoxic activity could improve future vaccine efficacy against such variants. [ABSTRACT FROM AUTHOR]

Small CD4-mimetic compound (CD4mc), which inhibits the interaction between gp120 with CD4, acts as an entry inhibitor and induces structural changes in the HIV-1 envelope glycoprotein trimer (Env) through its insertion within the Phe43 cavity of gp120. We recently developed YIR-821, a novel CD4mc, that has potent antiviral activity and lower toxicity than the prototype NBD-556. To assess the possibility of clinical application of YIR-821, we tested its antiviral activity using a panel of HIV-1 pseudoviruses from different subtypes. YIR-821 displayed entry inhibitor activity against 53.5% (21/40) of the pseudoviruses tested and enhanced neutralization mediated by coreceptor binding site (CoRBS) antibodies in 50% (16/ 32) of these. Furthermore, when we assessed the antiviral effects using a panel of pseudoviruses and autologous plasma IgG, enhancement of antibody-mediated neutralization activity was observed for 48% (15/31) of subtype B strains and 51% (28/55) of non-B strains. The direct antiviral activity of YIR-821 as an entry inhibitor was observed in 53% of both subtype B (27/51) and non-B subtype (40/75) pseudoviruses. Enhancement of antibody-dependent cellular cytotoxicity was also observed with YIR-821 for all six selected clinical isolates, as well as for the transmitted/founder (T/F) CH58 virus-infected cells. The sequence diversity in the CD4 binding site as well as other regions, such as the gp120 inner domain layers or gp41, may be involved in the multiple mechanisms related to the sensitive/resistant phenotype of the virus to YIR-821. Our findings may facilitate the clinical application of YIR-821.IMPORTANCE Small CD4-mimetic compound (CD4mc) interacts with the Phe43 cavity and triggers conformational changes, enhancing antibody-mediated neutralization and antibody-dependent cellular cytotoxicity (ADCC). Here, we evaluated the effect of YIR-821, a novel CD4mc, against clinical isolates, including both subtype B and non-B subtype viruses. Our results confirm the desirable properties of YIR-821, which include entry inhibition, enhancement of IgG-neutralization, binding, and ADCC, in addition to low toxicity and long half-life in a rhesus macaque model, that might facilitate the clinical application of this novel CD4mc. Our observation of primary viruses that are resistant to YIR-821 suggests that further development of CD4mcs with different structural properties is required. [ABSTRACT FROM AUTHOR]