Factors Associated with Neutralizing Antibody Responses following 2-Dose and 3rd Booster Monovalent COVID-19 Vaccination in Japanese People Living with HIV.

People living with HIV (PLWH) could be at risk of blunted immune responses to COVID-19 vaccination. We investigated factors associated with neutralizing antibody (NAb) responses against SARS-CoV-2 and variants of concern (VOCs), following two-dose and third booster monovalent COVID-19 mRNA vaccination in Japanese PLWH. NAb titers were assessed in polyclonal IgG fractions by lentiviral-based pseudovirus assays. Overall, NAb titers against Wuhan, following two-dose vaccination, were assessed in 82 PLWH on treatment, whereby 17/82 (20.73%) were classified as low-NAb participants. Within the low-NAb participants, the third booster vaccination enhanced NAb titers against Wuhan and VOCs, albeit to a significantly lower magnitude than the rest. In the multivariate analysis, NAb titers against Wuhan after two-dose vaccination correlated with age and days since vaccination, but not with CD4+ count, CD4+/CD8+ ratio, and plasma high-sensitivity C-Reactive protein (hsCRP). Interestingly, an extended analysis within age subgroups revealed NAb titers to correlate positively with the CD4+ count and negatively with plasma hsCRP in younger, but not older, participants. In conclusion, a third booster vaccination substantially enhances NAb titers, but the benefit may be suboptimal in subpopulations of PLWH exhibiting low titers at baseline. Considering clinical and immune parameters could provide a nuanced understanding of factors associated with vaccine responses in PLWH.

HIV-Tocky system to visualize proviral expression dynamics.

Determinants of HIV-1 latency establishment are yet to be elucidated. HIV reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model, HIV-Timer of cell kinetics and activity (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allows, for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies.

CD8+ T cell memory induced by successive SARS-CoV-2 mRNA vaccinations is characterized by shifts in clonal dominance.

mRNA vaccines against the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) elicit strong T cell responses. However, a clonal-resolution analysis of T cell responses to mRNA vaccination has not been performed. Here, we temporally track the CD8+ T cell repertoire in individuals who received three shots of the BNT162b2 mRNA vaccine through longitudinal T cell receptor sequencing with peptide-human leukocyte antigen (HLA) tetramer analysis. We demonstrate a shift in T cell responses between the clonotypes with different kinetics: from early responders that expand rapidly after the first shot to main responders that greatly expand after the second shot. Although the main responders re-expand after the third shot, their clonal diversity is skewed, and newly elicited third responders partially replace them. Furthermore, this shift in clonal dominance occurs not only between, but also within, clonotypes specific for spike epitopes. Our study will be a valuable resource for understanding vaccine-induced T cell responses in general.

Evaluation of Neutralizing Activity against Omicron Subvariants in BA.5 Breakthrough Infection and Three-Dose Vaccination Using a Novel Chemiluminescence-Based, Virus-Mediated Cytopathic Assay.

Neutralizing potency of humoral immune responses induced by prior infection or vaccination is vital for protecting of individuals and population against severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). However, the emergence of viral variants that can evade neutralization by vaccine- or infection-induced immunity is a significant public health threat and requires continuous monitoring. Here, we have developed a novel scalable chemiluminescence-based assay for assessing SARS-CoV-2-induced cytopathic effect to quantify the neutralizing activity of antisera. The assay leverages the correlation between host cell viability and ATP levels in culture to measure the cytopathic effect on target cells induced by clinically isolated, replication-competent, authentic SARS-CoV-2. With this assay, we demonstrate that the recently arisen Omicron subvariants BQ.1.1 and XBB.1 display a significant decrease in sensitivity to neutralization by antibodies elicited from breakthrough infections with Omicron BA.5 and from receipt of three doses of mRNA vaccines. Thus, this scalable neutralizing assay provides a useful platform to assess the potency of acquired humoral immunity against newly emerging SARS-CoV-2 variants. IMPORTANCE The ongoing global pandemic of SARS-CoV-2 has emphasized the importance of neutralizing immunity in protecting individuals and populations against severe respiratory illness. In light of the emergence of viral variants with the potential to evade immunity, continuous monitoring is imperative. A virus plaque reduction neutralization test (PRNT) is a "gold standard" assay for analyzing neutralizing activity for authentic viruses that form plaques, like influenza virus, dengue virus, and SARS-CoV-2. However, this method is labor intensive and is not efficient for performing large-scale neutralization assays on patient specimens. The assay system established in this study allows for the detection of a patient's neutralizing activity by simply adding an ATP detection reagent, providing a simple evaluation system for neutralizing activity of antisera as an alternative to the plaque reduction method. Our extended analysis of the Omicron subvariants highlights their increasing capability to evade neutralization by both vaccine- and infection-induced humoral immunity.

Association of Clostridium butyricum Therapy Using the Live Bacterial Product CBM588 with the Survival of Patients with Lung Cancer Receiving Chemoimmunotherapy Combinations.

The gut microbiota has emerged as a key regulator of immune checkpoint inhibitor (ICI) efficacy. Therapeutic approaches aimed at manipulating the microbiota through targeted reconstitution to enhance cancer treatment outcomes have garnered considerable attention. A single live microbial biotherapeutic bacterium, Clostridium butyricum MIYAIRI 588 strain (CBM588), has been shown to enhance the effects of ICI monotherapy in patients with advanced lung cancer. However, whether CBM588 affects the outcomes of chemoimmunotherapy combinations in lung cancer remains unknown. We hypothesized that CBM588 augments the effect of chemoimmunotherapy combinations and restores diminished effectiveness in patients with non-small cell lung cancer (NSCLC) receiving dysbiosis-inducing drugs. To validate this hypothesis, we retrospectively analyzed 106 patients with stage IV or recurrent metastatic NSCLC consecutively treated with chemoimmunotherapy combinations. A survival analysis was performed employing univariate and multivariate Cox proportional hazard models with inverse probability of treatment weighting (IPTW) using propensity scores. Forty-five percent of patients received Clostridium butyricum therapy. CBM588 significantly extended overall survival in patients with NSCLC receiving chemoimmunotherapy. The favorable impact of CBM588 on the efficacy of chemoimmunotherapy combinations varied based on tumor-programmed cell death ligand 1 (PD-L1) expression. The survival benefit of CBM588 in the PD-L1 <1% cohort was higher than that in the PD-L1 1-49% and PD-L1 ≥ 50% cohorts. Furthermore, CBM588 was associated with improved overall survival in patients receiving proton pump inhibitors and/or antibiotics. CBM588-induced manipulation of the commensal microbiota holds the potential to enhance the efficacy of chemoimmunotherapy combinations, warranting further exploration of the synergy between CBM588 and immunotherapy.

Symbiotic bacteria-dependent expansion of MR1-reactive T cells causes autoimmunity in the absence of Bcl11b.

MHC class I-related protein 1 (MR1) is a metabolite-presenting molecule that restricts MR1-reactive T cells including mucosal-associated invariant T (MAIT) cells. In contrast to MAIT cells, the function of other MR1-restricted T cell subsets is largely unknown. Here, we report that mice in which a T cell-specific transcription factor, B-cell lymphoma/leukemia 11B (Bcl11b), was ablated in immature thymocytes (Bcl11b∆iThy mice) develop chronic inflammation. Bcl11b∆iThy mice lack conventional T cells and MAIT cells, whereas CD4+IL-18R+ αß T cells expressing skewed Traj33 (Jα33)+ T cell receptors (TCR) accumulate in the periphery, which are necessary and sufficient for the pathogenesis. The disorders observed in Bcl11b∆iThy mice are ameliorated by MR1-deficiency, transfer of conventional T cells, or germ-free conditions. We further show the crystal structure of the TCR expressed by Traj33+ T cells expanded in Bcl11b∆iThy mice. Overall, we establish that MR1-reactive T cells have pathogenic potential.

Dissecting Naturally Arising Amino Acid Substitutions at Position L452 of SARS-CoV-2 Spike.

Mutations at spike protein L452 are recurrently observed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including omicron lineages. It remains elusive how amino acid substitutions at L452 are selected in VOC. Here, we characterized all 19 possible mutations at this site and revealed that five mutants expressing the amino acids Q, K, H, M, and R gained greater fusogenicity and pseudovirus infectivity, whereas other mutants failed to maintain steady-state expression levels and/or pseudovirus infectivity. Moreover, the five mutants showed decreased sensitivity toward neutralization by vaccine-induced antisera and conferred escape from T cell recognition. Contrary to expectations, sequence data retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) revealed that the naturally occurring L452 mutations were limited to Q, M, and R, all of which can arise from a single nucleotide change. Collectively, these findings highlight that the codon base change mutational barrier is a prerequisite for amino acid substitutions at L452, in addition to the phenotypic advantages of viral fitness and decreased sensitivity to host immunity. IMPORTANCE In a span of less than 3 years since the declaration of the coronavirus pandemic, numerous SARS-CoV-2 variants of concern have emerged all around the globe, fueling a surge in the number of cases and deaths that caused severe strain on the health care system. A major concern is whether viral evolution eventually promotes greater fitness advantages, transmissibility, and immune escape. In this study, we addressed the differential effect of amino acid substitutions at a frequent mutation site, L452 of SARS-CoV-2 spike, on viral antigenic and immunological profiles and demonstrated how the virus evolves to select one amino acid over the others to ensure better viral infectivity and immune evasion. Identifying such virus mutation signatures could be crucial for the preparedness of future interventions to control COVID-19.

The SARS-CoV-2 Omicron BA.1 spike G446S mutation potentiates antiviral T-cell recognition.

Although the Omicron variant of the SARS-CoV-2 virus shows resistance to neutralizing antibody, it retains susceptibility to the cellular immune response. Here we characterize vaccine-induced T cells specific for various SARS-CoV-2 variants and identified HLA-A*24:02-restricted CD8+ T cells that strongly suppress Omicron BA.1 replication in vitro. Mutagenesis analyses revealed that a G446S mutation, located just outside the N-terminus of the cognate epitope, augmented TCR recognition of this variant. In contrast, no enhanced suppression of replication is observed against cells infected with the prototype, Omicron BA.2, and Delta variants that express G446. The enhancing effect of the G446S mutation is lost when target cells are treated with inhibitors of tripeptidyl peptidase II, a protein that mediates antigen processing. These ex vivo analysis and in vitro results demonstrate that the G446S mutation in the Omicron BA.1 variant affects antigen processing/presentation and potentiates antiviral activity by vaccine-induced T cells, leading to enhanced T cell recognition towards emerging variants.

The SARS-CoV-2 Lambda variant exhibits enhanced infectivity and immune resistance.

SARS-CoV-2 Lambda, a variant of interest, has spread in some South American countries; however, its virological features and evolutionary traits remain unclear. In this study, we use pseudoviruses and reveal that the spike protein of the Lambda variant is more infectious than that of other variants due to the T76I and L452Q mutations. The RSYLTPGD246-253N mutation, a unique 7-amino acid deletion in the N-terminal domain of the Lambda spike protein, is responsible for evasion from neutralizing antibodies and further augments antibody-mediated enhancement of infection. Although this mutation generates a nascent N-linked glycosylation site, the additional N-linked glycan is dispensable for the virological property conferred by this mutation. Since the Lambda variant has dominantly spread according to the increasing frequency of the isolates harboring the RSYLTPGD246-253N mutation, our data suggest that the RSYLTPGD246-253N mutation is closely associated with the substantial spread of the Lambda variant in South America.

Resistance of SARS-CoV-2 variants to neutralization by antibodies induced in convalescent patients with COVID-19.

Administration of convalescent plasma or neutralizing monoclonal antibodies (mAbs) is a potent therapeutic option for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, SARS-CoV-2 variants with mutations in the spike protein have emerged in many countries. To evaluate the efficacy of neutralizing antibodies induced in convalescent patients against emerging variants, we isolate anti-spike mAbs from two convalescent COVID-19 patients infected with prototypic SARS-CoV-2 by single-cell sorting of immunoglobulin-G-positive (IgG+) memory B cells. Anti-spike antibody induction is robust in these patients, and five mAbs have potent neutralizing activities. The efficacy of most neutralizing mAbs and convalescent plasma samples is maintained against B.1.1.7 and mink cluster 5 variants but is significantly decreased against variants B.1.351 from South Africa and P.1 from Brazil. However, mAbs with a high affinity for the receptor-binding domain remain effective against these neutralization-resistant variants. Rapid spread of these variants significantly impacts antibody-based therapies and vaccine strategies against SARS-CoV-2.

SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity.

Many SARS-CoV-2 variants with naturally acquired mutations have emerged. These mutations can affect viral properties such as infectivity and immune resistance. Although the sensitivity of naturally occurring SARS-CoV-2 variants to humoral immunity has been investigated, sensitivity to human leukocyte antigen (HLA)-restricted cellular immunity remains largely unexplored. Here, we demonstrate that two recently emerging mutations in the receptor-binding domain of the SARS-CoV-2 spike protein, L452R (in B.1.427/429 and B.1.617) and Y453F (in B.1.1.298), confer escape from HLA-A24-restricted cellular immunity. These mutations reinforce affinity toward the host entry receptor ACE2. Notably, the L452R mutation increases spike stability, viral infectivity, viral fusogenicity, and thereby promotes viral replication. These data suggest that HLA-restricted cellular immunity potentially affects the evolution of viral phenotypes and that a further threat of the SARS-CoV-2 pandemic is escape from cellular immunity.

Trehalose diamide glycolipids augment antigen-specific antibody responses in a Mincle-dependent manner.

Many studies have investigated how trehalose glycolipid structures can be modified to improve their Macrophage inducible C-type lectin (Mincle)-mediated adjuvanticity. However, in all instances, the ester-linkage of α,ά-trehalose to the lipid of choice remained. We investigated how changing this ester-linkage to an amide influences Mincle signalling and agonist activity and demonstrated that Mincle tolerates this functional group change. In in vivo vaccination studies in murine and ovine model systems, using OVA or Mannheimia haemolytica and Mycoplasma ovipneumoniae as vaccine antigens, respectively, it was demonstrated that a representative trehalose diamide glycolipid was able to enhance antibody-specific immune responses. Notably, IgG titres against M. ovipneumoniae were significantly greater when using trehalose dibehenamide (A-TDB) compared to trehalose dibehenate (TDB). This is particularly important as infection with M. ovipneumoniae predisposes sheep to pneumonia.

The effects of 5-OP-RU stereochemistry on its stability and MAIT-MR1 axis.

Mucosal-associated invariant T (MAIT) cells are an abundant subset of innate-like T lymphocytes. MAIT cells are activated by microbial riboflavin-derived antigens, such as 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), when presented by the major histocompatibility complex (MHC) class I-related protein (MR1). We have synthesized all stereoisomers of 5-OP-RU to investigate the effects of its stereochemistry on the MR1-dependent MAIT cell activation and MR1 upregulation. The analysis of MAIT cell activation by these 5-OP-RU isomers revealed that the stereocenters at the 2'- and 3'-OH groups in the ribityl tail are crucial for the recognition of MAIT-TCR, whereas that of 4'-OH group does not significantly affect the regulation of MAIT cell activity. Furthermore, kinetic analysis of complex formation between the ligands and MR1 suggested that 5-OP-RU forms a covalent bond to MR1 in cells within 1 hour. These findings provide guidelines for designing ligands that regulate MAIT cell functions.

Agonistic or antagonistic mucosal-associated invariant T (MAIT) cell activity is determined by the 6-alkylamino substituent on uracil MR1 ligands.

Mucosal-associated invariant T (MAIT) are a subset of innate-like T cells that are activated by uracil ligands presented by MR1. For the first time, we demonstrate that changes to the 6-aminoalkyl chain on uracil agonist 5-OP-RU can determine agonistic or antagonistic MAIT cell activity. Insomuch, a simplified agonist with a functional profile similar to 5-OP-RU, and a new structural class of antagonist that exhibits similar activity to known MAIT cell antagonist Ac-6-FP, were identified.

In vivo delivery of an exogenous molecule into murine T lymphocytes using a lymphatic drug delivery system combined with sonoporation.

Physical delivery of exogenous molecules into lymphocytes is extremely challenging because conventional methods have notable limitations. Here, we evaluated the potential use of acoustic liposomes (ALs) and sonoporation to deliver exogenous molecules into lymphocytes within a lymph node (LN). MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice, which show systemic LN swelling, were used as the model system. After direct injection into the subiliac LN, a solution containing both ALs and TOTO-3 fluorophores (molecular weight: 1355) was able to reach the downstream proper axillary LN (PALN) via the lymphatic vessels (LVs). This led to the accumulation of a high concentration of TOTO-3 fluorophores and ALs in the lymphatic sinuses of the PALN, where a large number of lymphocytes were densely packed. Exposure of the PALN to >1.93 W/cm2 of 970-kHz ultrasound allowed the solution to extravasate into the parenchyma and reach the large number of lymphocytes in the sinuses. Flow cytometric analysis showed that TOTO-3 molecules were delivered into 0.49 ± 0.23% of CD8+7AAD- cytotoxic T lymphocytes. Furthermore, there was no evidence of tissue damage. Thus, direct administration of drugs into LVs combined with sonoporation can improve the delivery of exogenous molecules into primary lymphocytes. This technique could become a novel approach to immunotherapy.

Interstitial-resident memory CD8+ T cells sustain frontline epithelial memory in the lung.

Populations of CD8+ lung-resident memory T (TRM) cells persist in the interstitium and epithelium (airways) following recovery from respiratory virus infections. While it is clear that CD8+ TRM cells in the airways are dynamically maintained via the continuous recruitment of new cells, there is a vigorous debate about whether tissue-circulating effector memory T (TEM) cells are the source of these newly recruited cells. Here we definitively demonstrate that CD8+ TRM cells in the lung airways are not derived from TEM cells in the circulation, but are seeded continuously by TRM cells from the lung interstitium. This process is driven by CXCR6 that is expressed uniquely on TRM cells but not TEM cells. We further demonstrate that the lung interstitium CD8+ TRM cell population is also maintained independently of TEM cells via a homeostatic proliferation mechanism. Taken together, these data show that lung memory CD8+ TRM cells in the lung interstitium and airways are compartmentally separated from TEM cells and clarify the mechanisms underlying their maintenance.

The effect of MR1 ligand glyco-analogues on mucosal-associated invariant T (MAIT) cell activation.

Mucosal-associated invariant T (MAIT) cells are a subset of recently identified innate-like T lymphocytes that appear to play an important role in many pathologies ranging from viral and bacterial infection, to autoimmune disorders and cancer. MAIT cells are activated via the presentation of ligands by MR1 on antigen presenting cells to the MAIT T cell receptor (TCR), however few studies have explored the effects of systematic changes to the ligand structure on MR1 binding and MAIT cell activation. Herein, we report on the first study into the effects of changes to the sugar motif in the known MAIT cell agonists 7-hydroxy-6-methyl-8-d-ribityllumazine (RL-6-Me-7-OH) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU). Tetramer staining of MAIT cells revealed that the absence of the 2'-hydroxy group on the sugar backbone of lumazines improved MR1-MAIT TCR binding, which could be rationalised using computational docking studies. Although none of the lumazines activated MAIT cells, all 5-OP-RU analogues showed significant MAIT cell activation, with several analogues exhibiting comparable activity to 5-OP-RU. Docking studies with the 5-OP-RU analogues revealed different interactions between the sugar backbone and MR1 and the MAIT TCR compared to those observed for the lumazines and confirmed the importance of the 2'-hydroxy group for ligand binding and activity. Taken together, this information will assist in the development of future potent agonists and antagonists of MAIT cells.

Regulation of membrane phospholipid asymmetry by Notch-mediated flippase expression controls the number of intraepithelial TCRαß+CD8αα+ T cells.

Intestinal intraepithelial lymphocytes (IELs) expressing CD8αα on αß T cells (TCRαß+CD8αα+ IELs) have suppressive capabilities in enterocolitis, but the mechanism that maintains homeostasis and cell number is not fully understood. Here, we demonstrated that the number of TCRαß+CD8αα+ IELs was severely reduced in mice lacking recombination signal binding protein for immunoglobulin kappa J region (Rbpj) or Notch1 and Notch2 in T cells. Rbpj-deficient TCRαß+CD8αα+ IELs expressed low levels of Atp8a2, which encodes a protein with flippase activity that regulates phospholipid asymmetry of plasma membrane such as flipping phosphatidylserine in the inner leaflet of plasma membrane. Rbpj-deficient TCRαß+CD8αα+ IELs cannot maintain phosphatidylserine in the inner leaflet of the plasma membrane. Furthermore, depletion of intestinal macrophages restored TCRαß+CD8αα+ IELs in Rbpj-deficient mice, suggesting that exposure of phosphatidylserine on the plasma membrane in Rbpj-deficient TCRαß+CD8αα+ IELs acts as an "eat-me" signal. Together, these results revealed that Notch-Atp8a2 is a fundamental regulator for IELs and highlighted that membrane phospholipid asymmetry controlled by Notch-mediated flippase expression is a critical determinant in setting or balancing the number of TCRαß+CD8αα+ IELs.

Dectin-1 intracellular domain determines species-specific ligand spectrum by modulating receptor sensitivity.

C-type lectin receptors (CLRs) comprise a large family of immunoreceptors that recognize polysaccharide ligands exposed on pathogen surfaces and are conserved among mammals. However, interspecies differences in their ligand spectrums are not fully understood. Dectin-1 is a well-characterized CLR that recognizes ß-glucan. We report here that seaweed-derived fucan activates cells expressing human Dectin-1 but not mouse Dectin-1. Low-valency ß-glucan components within fucan appeared to be responsible for this activation, as the ligand activity was eliminated by ß-glucanase treatment. The low-valency ß-glucan laminarin also acted as an agonist for human Dectin-1 but not for mouse Dectin-1, whereas the high-valency ß-glucan curdlan activated both human and mouse Dectin-1. Reciprocal mutagenesis analysis revealed that the ligand-binding domain of human Dectin-1 does not determine its unique sensitivity to low-valency ß-glucan. Rather, we found that its intracellular domain renders human Dectin-1 reactive to low-valency ß-glucan ligand. Substitution with two amino acids, Glu2 and Pro5, located in the human Dectin-1 intracellular domain was sufficient to confer sensitivity to low-valency ß-glucan in mouse Dectin-1. Conversely, the introduction of mouse-specific amino acids, Lys2 and Ser5, to human Dectin-1 reduced the reactivity to low-valency ß-glucan. Indeed, low-valency ligands induced a set of proinflammatory genes in human but not mouse dendritic cells. These results suggest that the intracellular domain, not ligand-binding domain, of Dectin-1 determines the species-specific ligand profile.