Structural basis for cross-group recognition of an influenza virus hemagglutinin antibody that targets postfusion stabilized epitope.

Plasticity of influenza virus hemagglutinin (HA) conformation increases an opportunity to generate conserved non-native epitopes with unknown functionality. Here, we have performed an in-depth analysis of human monoclonal antibodies against a stem-helix region that is occluded in native prefusion yet exposed in postfusion HA. A stem-helix antibody, LAH31, provided IgG Fc-dependent cross-group protection by targeting a stem-helix kinked loop epitope, with a unique structure emerging in the postfusion state. The structural analysis and molecular modeling revealed key contact sites responsible for the epitope specificity and cross-group breadth that relies on somatically mutated light chain. LAH31 was inaccessible to the native prefusion HA expressed on cell surface; however, it bound to the HA structure present on infected cells with functional linkage to the Fc-mediated clearance. Our study uncovers a novel non-native epitope that emerges in the postfusion HA state, highlighting the utility of this epitope for a broadly protective antigen design.

CD62L expression marks SARS-CoV-2 memory B cell subset with preference for neutralizing epitopes.

Severe acute respiratory syndrome coronavirus 2-neutralizing antibodies primarily target the spike receptor binding domain (RBD). However, B cell antigen receptors (BCRs) on RBD-binding memory B (Bmem) cells have variation in the neutralizing activities. Here, by combining single Bmem cell profiling with antibody functional assessment, we dissected the phenotype of Bmem cell harboring the potently neutralizing antibodies in coronavirus disease 2019 (COVID-19)-convalescent individuals. The neutralizing subset was marked by an elevated CD62L expression and characterized by distinct epitope preference and usage of convergent VH (variable region of immunoglobulin heavy chain) genes, accounting for the neutralizing activities. Concordantly, the correlation was observed between neutralizing antibody titers in blood and CD62L+ subset, despite the equivalent RBD binding of CD62L+ and CD62L- subset. Furthermore, the kinetics of CD62L+ subset differed between the patients who recovered from different COVID-19 severities. Our Bmem cell profiling reveals the unique phenotype of Bmem cell subset that harbors potently neutralizing BCRs, advancing our understanding of humoral protection.

Post-fusion influenza vaccine adjuvanted with SA-2 confers heterologous protection via Th1-polarized, non-neutralizing antibody responses.

Development of a universal influenza vaccine that can provide robust and long-lasting protection against heterologous infections is a global public health priority. A variety of vaccine antigens are designed to increase the antigenicity of conserved epitopes to elicit cross-protective antibodies that often lack virus-neutralizing activity. Given the contribution of antibody effector functions to cross-protection, adjuvants need to be added to modulate antibody effector functions as well as to enhance antibody quantity. We previously showed that post-fusion influenza vaccine antigens elicit non-neutralizing but cross-protective antibodies against conserved epitopes. Here, using a murine model, we comparably assessed the adjuvanticity of the newly developed SA-2 adjuvant containing a synthetic TLR7 agonist DSP-0546 and squalene-based MF59 analog as representative Th1- or Th2-type adjuvants, respectively. Both types of adjuvants in the post-fusion vaccine comparably enhanced cross-reactive IgG titers against heterologous strains. However, only SA-2 skewed the IgG subclass into the IgG2c subclass in association to its Th1-polarizing nature. SA-2-enhanced IgG2c responses exhibited antibody-dependent cellular cytotoxicity against heterologous virus strains, without cross-neutralizing activity. Eventually, the SA-2-adjuvanted vaccination provided protection against lethal infection by heterologous H3N2 and H1N1 viruses. Together, we conclude that the combination with a SA-2 is advantageous for enhancing the cross-protective capability of post-fusion HA vaccines that elicit non-neutralizing IgG antibodies.

Gene expression analysis during the conversion from a viable but nonculturable to culturable state in Vibrio cholerae.

We previously reported that Vibrio cholerae in a viable but non-culturable (VBNC) state can be converted to a culturable state by treatment with catalase. This finding enabled us to develop an assay system to observe the time course of the conversion from VBNC to culturable in V. cholerae. VBNC cells began to convert to culturable cells as early as 2 h after catalase supplementation. Gene expression in VBNC cells during catalase treatment was analyzed using RNA microarray. Many ribosomal DNA genes were stimulated 6 h post catalase exposure, suggesting that the conversion-driving signal started prior to 6 h. Focusing on the period prior to cell proliferation, we found that 16 genes might be involved in the conversion mechanism in V. cholerae, and they showed enhanced expression at 2 h and 4 h after catalase addition. These upregulated genes included phage shock proteins (pspA, B, and C), alternative sigma factor (rpoE) and its negative regulator (rseA), cobW C terminal domain-containing protein, damage-inducible helicase (dinG), cholerae toxin secretion protein epsM, HTH-type transcription regulator (iscR), mechanosensitive ion channel family protein, anthranilate synthase component I, fructose-specific IIBC component, molybdenum import ATP-binding protein (modC), LysE family translocator, putative organic hydroperoxide resistance protein, and a hypothetical protein. This study identified genes involved in the catalase-induced conversion of V. cholerae VBNC cells to a culturable state and provided valuable insights into the mechanisms involved in the conversion process.

SARS-CoV-2-specific CD4+ T cell longevity correlates with Th17-like phenotype.

Determinants of memory T cell longevity following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain unknown. In addition, phenotypes associated with memory T cell longevity, antibody titers, and disease severity are incompletely understood. Here, we longitudinally analyzed SARS-CoV-2-specific T cell and antibody responses of a unique cohort with similar numbers of mild, moderate, and severe coronavirus disease 2019 cases. The half-lives of CD4+ and CD8+ T cells were longer than those of antibody titers and showed no clear correlation with disease severity. When CD4+ T cells were divided into Th1-, Th2-, Th17-, and Tfh-like subsets, the Th17-like subset showed a longer half-life than other subsets, indicating that Th17-like cells are most closely correlated with T cell longevity. In contrast, Th2- and Tfh-like T cells were more closely correlated with antibody titers than other subsets. These results suggest that distinct CD4+ T cell subsets are associated with longevity and antibody responses.

Distinct immune cell dynamics correlate with the immunogenicity and reactogenicity of SARS-CoV-2 mRNA vaccine.

Two doses of Pfizer/BioNTech BNT162b2 mRNA vaccine elicit robust severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing antibodies with frequent adverse events. Here, by applying a high-dimensional immune profiling on 92 vaccinees, we identify six vaccine-induced immune dynamics that correlate with the amounts of neutralizing antibodies, the severity of adverse events, or both. The early dynamics of natural killer (NK)/monocyte subsets (CD16+ NK cells, CD56high NK cells, and non-classical monocytes), dendritic cell (DC) subsets (DC3s and CD11c- Axl+ Siglec-6+ [AS]-DCs), and NKT-like cells are revealed as the distinct cell correlates for neutralizing-antibody titers, severity of adverse events, and both, respectively. The cell correlates for neutralizing antibodies or adverse events are consistently associated with elevation of interferon gamma (IFN-γ)-inducible chemokines, but the chemokine receptors CCR2 and CXCR3 are expressed in distinct manners between the two correlates: vaccine-induced expression on the neutralizing-antibody correlate and constitutive expression on the adverse-event correlate. The finding may guide vaccine strategies that balance immunogenicity and reactogenicity.

SARS-CoV-2 Omicron-neutralizing memory B cells are elicited by two doses of BNT162b2 mRNA vaccine.

Multiple SARS-CoV-2 variants have mutations in the spike receptor binding domain (RBD) with potential to evade neutralizing antibody. In particular, the Beta and Omicron variants escape from antibody neutralizing activity in those who received two doses of BNT162b2 mRNA vaccine. Nonetheless, boosting with a third vaccine dose or by breakthrough infection improves the overall breadth of the neutralizing antibodies, but the mechanism remains unclear. Here, we longitudinally profiled the cellular composition of RBD-binding memory B cell subsets and their antibody binding and neutralizing activity against SARS-CoV-2 variants after the second dose of mRNA vaccine. Two doses of the mRNA vaccine elicited plasma neutralizing antibodies with a limited activity against Beta and Omicron but induced an expanded antibody breadth overtime, up to 4.9 months after vaccination. In contrast, more than one-third of RBD-binding IgG+ memory B cells with a resting phenotype initially bound the Beta and Omicron variants and steadily increased the B cell receptor breadth overtime. As a result, a fraction of the resting memory B cell subset secreted Beta and Omicron-neutralizing antibody when stimulated in vitro. The neutralizing breadth of the resting memory B cell subset helps us understand the prominent recall of Omicron-neutralizing antibodies after an additional booster or breakthrough infection in fully vaccinated individuals.

Temporal maturation of neutralizing antibodies in COVID-19 convalescent individuals improves potency and breadth to circulating SARS-CoV-2 variants.

Antibody titers against SARS-CoV-2 slowly wane over time. Here, we examined how time affects antibody potency. To assess the impact of antibody maturation on durable neutralizing activity against original SARS-CoV-2 and emerging variants of concern (VOCs), we analyzed receptor binding domain (RBD)-specific IgG antibodies in convalescent plasma taken 1-10 months after SARS-CoV-2 infection. Longitudinal evaluation of total RBD IgG and neutralizing antibody revealed declining total antibody titers but improved neutralization potency per antibody to original SARS-CoV-2, indicative of antibody response maturation. Neutralization assays with authentic viruses revealed that early antibodies capable of neutralizing original SARS-CoV-2 had limited reactivity toward B.1.351 (501Y.V2) and P.1 (501Y.V3) variants. Antibodies from late convalescents exhibited increased neutralization potency to VOCs, suggesting persistence of cross-neutralizing antibodies in plasma. Thus, maturation of the antibody response to SARS-CoV-2 potentiates cross-neutralizing ability to circulating variants, suggesting that declining antibody titers may not be indicative of declining protection.

Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae.

Many pathogenic bacteria, including Escherichia coli and Vibrio cholerae, can become viable but nonculturable (VBNC) following exposure to specific stress conditions. Corynebacterium diphtheriae, a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that C. diphtheriae can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC C. diphtheriae were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions (culturable, VBNC, VBNC after the addition of catalase, and resuscitated cells). This is the first study to demonstrate that C. diphtheriae can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC C. diphtheriae, and its environmental survival strategy.

Myeloid cell dynamics correlating with clinical outcomes of severe COVID-19 in Japan.

An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19). However, data regarding myeloid cell expansion have been collected in Europe, where the mortality rate by COVID-19 is greater than those in other regions including Japan. Thus, characteristics of COVID-19-induced myeloid cell subsets remain largely unknown in the regions with low mortality rates. Here, we analyzed cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis, using blood samples from Japanese COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, but not other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Contrary to previous studies in Europe, this subset selectively expanded in survivors of severe cases and subsided before discharge, but such transient expansion was not observed in non-survivors in Japanese cohort. Analysis of plasma cytokine/chemokine levels revealed positive correlation of PMN-MDSC frequencies with IL-8 levels, indicating the involvement of IL-8 on recruitment of PMN-MDSCs to peripheral blood following the onset of severe COVID-19. Our data indicate that transient expansion of the PMN-MDSC subset results in improved clinical outcome. Thus, this myeloid cell subset may be a predictor of prognosis in cases of severe COVID-19 in Japan.

An anti-perfringolysin O monoclonal antibody cross-reactive with streptolysin O protects against streptococcal toxic shock syndrome.

OBJECTIVE: Streptococcus pyogenes (Group A Streptococcus; GAS) causes a variety of infections that include life-threatening, severe invasive GAS infections, such as streptococcal toxic shock syndrome (STSS), with > 30% mortality rate, despite effective antibiotics and treatment options. STSS clinical isolates highly express streptolysin O (SLO), a member of a large family of pore-forming toxins called cholesterol-dependent cytolysins (CDCs). SLO is an important toxic factor for GAS and may be an effective therapeutic target for the treatment of STSS. Our aim was to identify a monoclonal antibody (mAb) that reacts with SLO and has therapeutic potential for STSS treatment. RESULTS: We focused on mAbs that had originally been established as neutralizing reagents to perfringolysin O (PFO), another member of the CDC family, as some cross-reactivity with SLO had been reported. Here, we confirmed cross-reactivity of an anti-PFO mAb named HS1 with SLO. In vitro analysis revealed that HS1 mAb sufficiently prevented human neutrophils from being killed by STSS clinical isolates. Furthermore, prophylactic and therapeutic injection of HS1 mAb into C57BL/6 mice significantly improved the survival rate following lethal infection with an STSS clinical isolate. These results highlight the therapeutic potential of HS1 mAb for STSS treatment.

Glycine plays a crucial role as a co-agonist of NMDA receptors in the neuronal circuit generating body movements in rat fetuses.

Neuronal circuits generating fetal movements in mammals are localized in the brainstem and the spinal cord. It has been shown that glycine plays an important role through the strychnine-sensitive glycine receptors in these circuits. However, the role of glycine as the NMDA receptor co-agonist in fetal period is not fully understood. In this study, we examined the contribution of glycine to the perinatal rat spinal circuit generating forelimb movements utilizing isolated brainstem-cervical-spinal-cord preparations. In late embryonic-days-preparations, spontaneous motor bursts related to forelimb movements (forelimb-movement-related bursts; FMRBs) and respiration-related activity were observed. In neonatal preparations, spontaneous FMRBs were not observed but periodic motor bursts resembling the FMRBs could be induced after bath application of strychnine (strychnine-induced motor bursts; SIMBs). Both FMRBs and SIMBs were blocked by either the NMDA receptor antagonist APV or the antagonists of the glycine binding site of NMDA receptors [5,7-dichlorokynurenic acid (DCKA) or L-689560]. Furthermore, these motor bursts were facilitated by the glycine uptake blocker sarcosine. This effect of sarcosine was blocked by DCKA. The findings indicate that glycine plays a crucial role as a NMDA receptor co-agonist in generating spontaneous fetal motor activity before functioning as a classical inhibitory neurotransmitter in suppressing the fetal neuronal circuits.

Glycemic index and glucose utilization of rice vermicelli in healthy subjects.

Glycemic index (GI) is an indicator of glucose absorption into the systemic circulation after ingestion of foods. However, the plasma glucose level is determined by not only the absorption of glucose, but also the disappearance of glucose which is regulated by the insulin response. The aim of this study was to estimate the values of GI and glucose utilization including the absorption and the disappearance of glucose (U(GLU)) in the rice vermicelli (RV) using the resulting glucose clearance (CL(GLU)). Fifteen healthy subjects participated in this study. Alterations in plasma glucose and insulin levels were determined after ingestion of a reference food (12.5, 25, 50, 75 g glucose) and the test foods (white rice, long grain rice and six RV products; 50 g available carbohydrate). Time-course changes in plasma glucose levels were analyzed using a simple kinetic model. The values of CL(GLU) were calculated using the resulting kinetic parameters. A standard curve in which the incremental area under the curve of plasma insulin levels (AUC(INS(ref))) was plotted against the resulting CL(GLU(ref)) was generated using the reference food. The values of GI and U(GLU) in the RV products were calculated from the observed clearances (CL(GLU)), and the predicted clearances (CL(GLU(predict))) respectively, which were estimated using the standard curve of AUC(INS(ref)) vs. CL(GLU(ref)). It was clarified that the values of U(GLU) in the RV products (20-57%) were lower than those of GI (35-62%).

Mannitol lowers fat digestibility and body fat accumulation in both normal and cecectomized rats.

Mannitol is a six-carbon sugar alcohol that is widely distributed in plants. Sugar alcohols are widely used in various food products because of their numerous beneficial health effects. The present study investigated the effects of mannitol consumption on digestion, large gut fermentation and nutrient metabolism in normal and cecectomized male Wistar rats. After 28 d of feeding with three kinds of diet containing 0, 4 or 8% mannitol, mannitol consumption dose-dependently lowered the digestibilities of crude fat and crude protein, the ratio of body fat accumulation to energy absorbed and the hepatic and serum triglyceride levels in normal rats. After 24 d of feeding with three kinds of diet comprising a control diet, a 5% mannitol-containing diet and a 5% fructooligosaccharides (FOS)-containing diet, mannitol lowered the digestibility of fat and the ratio of body fat accumulation to energy consumed and absorbed in cecectomized rats. On the other hand, FOS consumption had no effect on the accumulation of body fat, but lowered the digestibility of fat. FOS consumption greatly improved the accumulation of body ash in cecectomized rats. These results suggest that mannitol has a lowering effect on body fat accumulation, and further indicate that the cecum is not essential for the appearance of effects of mannitol on digestion, absorption and metabolism.

Rice gruel and rice starch reduce sorbitol-induced diarrhoea in cecectomized rats.

BACKGROUND/AIMS: Sorbitol (S), a widely used sugar substitute in sugar-free products, has shown intolerance manifested by abdominal pain, bloating and diarrhoea in children and adults. The nutritional and S-induced diarrhoea-preventive effect of rice gruel (RG) and rice starch (RS) was studied in cecectomized rats. METHODS: During the 41-day experimental period, control, RG or RS, S, and S+RG or RS-containing diets were provided in four consecutive phases. The diet compositions were changed depending on the conditions of the diarrhoea. Faecal appearances of S- and S+RG/RS-fed groups were observed daily and scored (diarrhoea = 2, mixed fae ces = 1, normal faeces = 0) to evaluate the degree of diarrhoea. RESULTS: The diarrhoeal scores were significantly (p < 0.05) decreased in all phases of the RG- or RS-fed group compared to the control group (S). Serum lipid and colonic organic acid concentrations were decreased while faecal bile acids and neutral sterol concentrations were increased in the RG-fed group compared to the control which were measured in the samples collected from the last phase of the experiment. CONCLUSION: These results indicate that RG and RS may be useful additives in S-containing food products to prevent the possible risk of diarrhoea in humans.