Conversion of monoclonal IgG to dimeric and secretory IgA restores neutralizing ability and prevents infection of Omicron lineages.

The emergence of Omicron lineages and descendent subvariants continues to present a severe threat to the effectiveness of vaccines and therapeutic antibodies. We have previously suggested that an insufficient mucosal immunoglobulin A (IgA) response induced by the mRNA vaccines is associated with a surge in breakthrough infections. Here, we further show that the intramuscular mRNA and/or inactivated vaccines cannot sufficiently boost the mucosal secretory IgA response in uninfected individuals, particularly against the Omicron variant. We thus engineered and characterized recombinant monomeric, dimeric, and secretory IgA1 antibodies derived from four neutralizing IgG monoclonal antibodies (mAbs 01A05, rmAb23, DXP-604, and XG014) targeting the receptor-binding domain of the spike protein. Compared to their parental IgG antibodies, dimeric and secretory IgA1 antibodies showed a higher neutralizing activity against different variants of concern (VOCs), in part due to an increased avidity. Importantly, the dimeric or secretory IgA1 form of the DXP-604 antibody significantly outperformed its parental IgG antibody, and neutralized the Omicron lineages BA.1, BA.2, and BA.4/5 with a 25- to 75-fold increase in potency. In human angiotensin converting enzyme 2 (ACE2) transgenic mice, a single intranasal dose of the dimeric IgA DXP-604 conferred prophylactic and therapeutic protection against Omicron BA.5. Thus, dimeric or secretory IgA delivered by nasal administration may potentially be exploited for the treatment and prevention of Omicron infection, thereby providing an alternative tool for combating immune evasion by the current circulating subvariants and, potentially, future VOCs.

Genetic and immunologic evaluation of children with inborn errors of immunity and severe or critical COVID-19.

BACKGROUND: Most severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals are asymptomatic or only exhibit mild disease. In about 10% of cases, the infection leads to hypoxemic pneumonia, although it is much more rare in children. OBJECTIVE: We evaluated 31 young patients aged 0.5 to 19 years who had preexisting inborn errors of immunity (IEI) but lacked a molecular diagnosis and were later diagnosed with coronavirus disease 2019 (COVID-19) complications. METHODS: Genetic evaluation by whole-exome sequencing was performed in all patients. SARS-CoV-2-specific antibodies, autoantibodies against type I IFN (IFN-I), and inflammatory factors in plasma were measured. We also reviewed COVID-19 disease severity/outcome in reported IEI patients. RESULTS: A potential genetic cause of the IEI was identified in 28 patients (90.3%), including mutations that may affect IFN signaling, T- and B-cell function, the inflammasome, and the complement system. From tested patients 65.5% had detectable virus-specific antibodies, and 6.8% had autoantibodies neutralizing IFN-I. Five patients (16.1%) fulfilled the diagnostic criteria of multisystem inflammatory syndrome in children. Eleven patients (35.4%) died of COVID-19 complications. All together, at least 381 IEI children with COVID-19 have been reported in the literature to date. Although many patients with asymptomatic or mild disease may not have been reported, severe presentation of COVID-19 was observed in 23.6% of the published cases, and the mortality rate was 8.7%. CONCLUSIONS: Young patients with preexisting IEI may have higher mortality than children without IEI when infected with SARS-CoV-2. Elucidating the genetic basis of IEI patients with severe/critical COVID-19 may help to develop better strategies for prevention and treatment of severe COVID-19 disease and complications in pediatric patients.

SARS-CoV-2-specific B- and T-cell immunity in a population-based study of young Swedish adults.

BACKGROUND: Young adults are now considered major spreaders of coronavirus disease 2019 (COVID-19) disease. Although most young individuals experience mild to moderate disease, there are concerns of long-term adverse health effects. The impact of COVID-19 disease and to which extent population-level immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exists in young adults remain unclear. OBJECTIVE: We conducted a population-based study on humoral and cellular immunity to SARS-CoV-2 and explored COVID-19 disease characteristics in young adults. METHODS: We invited participants from the Swedish BAMSE (Barn [Children], Allergy Milieu, Stockholm, Epidemiology) birth cohort (age 24-27 years) to take part in a COVID-19 follow-up. From 980 participants (October 2020 to June 2021), we here present data on SARS-CoV-2 receptor-binding domain-specific IgM, IgA, and IgG titers measured by ELISA and on symptoms and epidemiologic factors associated with seropositivity. Further, SARS-CoV-2-specific memory B- and T-cell responses were detected for a subpopulation (n = 108) by ELISpot and FluoroSpot. RESULTS: A total of 28.4% of subjects were seropositive, of whom 18.4% were IgM single positive. One in 7 seropositive subjects was asymptomatic. Seropositivity was associated with use of public transport, but not with sex, asthma, rhinitis, IgE sensitization, smoking, or body mass index. In a subset of representative samples, 20.7% and 35.0% had detectable SARS-CoV-2 specific B- and T-cell responses, respectively. B- and T-cell memory responses were clearly associated with seropositivity, but T-cell responses were also detected in 17.2% of seronegative subjects. CONCLUSIONS: Assessment of IgM and T-cell responses may improve population-based estimations of SARS-CoV-2 infection. The pronounced surge of both symptomatic and asymptomatic infections among young adults indicates that the large-scale vaccination campaign should be continued.

Inherited IFNAR1 Deficiency in a Child with Both Critical COVID-19 Pneumonia and Multisystem Inflammatory Syndrome.

BACKGROUND: Inborn errors of immunity (IEI) and autoantibodies to type I interferons (IFNs) underlie critical COVID-19 pneumonia in at least 15% of the patients, while the causes of multisystem inflammatory syndrome in children (MIS-C) remain elusive. OBJECTIVES: To detect causal genetic variants in very rare cases with concomitant critical COVID-19 pneumonia and MIS-C. METHODS: Whole exome sequencing was performed, and the impact of candidate gene variants was investigated. Plasma levels of cytokines, specific antibodies against the virus, and autoantibodies against type I IFNs were also measured. RESULTS: We report a 3-year-old child who died on day 56 of SARS-CoV-2 infection with an unusual clinical presentation, combining both critical COVID-19 pneumonia and MIS-C. We identified a large, homozygous loss-of-function deletion in IFNAR1, underlying autosomal recessive IFNAR1 deficiency. CONCLUSIONS: Our findings confirm that impaired type I IFN immunity can underlie critical COVID-19 pneumonia, while suggesting that it can also unexpectedly underlie concomitant MIS-C. Our report further raises the possibility that inherited or acquired dysregulation of type I IFN immunity might contribute to MIS-C in other patients.

X-Linked TLR7 Deficiency Underlies Critical COVID-19 Pneumonia in a Male Patient with Ataxia-Telangiectasia.

BACKGROUND: Coronavirus disease 2019 (COVID-19) exhibits a wide spectrum of clinical manifestations, ranging from asymptomatic to critical conditions. Understanding the mechanism underlying life-threatening COVID-19 is instrumental for disease prevention and treatment in individuals with a high risk. OBJECTIVES: We aimed to identify the genetic cause for critical COVID-19 pneumonia in a patient with a preexisting inborn error of immunity (IEI). METHODS: Serum levels of specific antibodies against the virus and autoantibodies against type I interferons (IFNs) were measured. Whole exome sequencing was performed, and the impacts of candidate gene variants were investigated. We also evaluated 247 ataxia-telangiectasia (A-T) patients in the Iranian IEI registry. RESULTS: We report a 7-year-old Iranian boy with a preexisting hyper IgM syndrome who developed critical COVID-19 pneumonia. IgM only specific COVID-19 immune response was detected but no autoantibodies against type I IFN were observed. A homozygous deleterious mutation in the ATM gene was identified, which together with his antibody deficiency, radiosensitivity, and neurological signs, established a diagnosis of A-T. Among the 247 A-T patients evaluated, 36 had SARS-CoV-2 infection, but all had mild symptoms or were asymptomatic except the index patient. A hemizygous deleterious mutation in the TLR7 gene was subsequently identified in the patient. CONCLUSIONS: We report a unique IEI patient with combined ATM and TLR7 deficiencies. The two genetic defects underlie A-T and critical COVID-19 in this patient, respectively.

Human serum from SARS-CoV-2-vaccinated and COVID-19 patients shows reduced binding to the RBD of SARS-CoV-2 Omicron variant.

BACKGROUND: The COVID-19 pandemic is caused by the betacoronavirus SARS-CoV-2. In November 2021, the Omicron variant was discovered and immediately classified as a variant of concern (VOC), since it shows substantially more mutations in the spike protein than any previous variant, especially in the receptor-binding domain (RBD). We analyzed the binding of the Omicron RBD to the human angiotensin-converting enzyme-2 receptor (ACE2) and the ability of human sera from COVID-19 patients or vaccinees in comparison to Wuhan, Beta, or Delta RBD variants. METHODS: All RBDs were produced in insect cells. RBD binding to ACE2 was analyzed by ELISA and microscale thermophoresis (MST). Similarly, sera from 27 COVID-19 patients, 81 vaccinated individuals, and 34 booster recipients were titrated by ELISA on RBDs from the original Wuhan strain, Beta, Delta, and Omicron VOCs. In addition, the neutralization efficacy of authentic SARS-CoV-2 wild type (D614G), Delta, and Omicron by sera from 2× or 3× BNT162b2-vaccinated persons was analyzed. RESULTS: Surprisingly, the Omicron RBD showed a somewhat weaker binding to ACE2 compared to Beta and Delta, arguing that improved ACE2 binding is not a likely driver of Omicron evolution. Serum antibody titers were significantly lower against Omicron RBD compared to the original Wuhan strain. A 2.6× reduction in Omicron RBD binding was observed for serum of 2× BNT162b2-vaccinated persons. Neutralization of Omicron SARS-CoV-2 was completely diminished in our setup. CONCLUSION: These results indicate an immune escape focused on neutralizing antibodies. Nevertheless, a boost vaccination increased the level of anti-RBD antibodies against Omicron, and neutralization of authentic Omicron SARS-CoV-2 was at least partially restored. This study adds evidence that current vaccination protocols may be less efficient against the Omicron variant.

Lactobacilli Downregulate Transcription Factors in Helicobacter pylori That Affect Motility, Acid Tolerance and Antimicrobial Peptide Survival.

Helicobacter pylori infection triggers inflammation that may lead to gastritis, stomach ulcers and cancer. Probiotic bacteria, such as Lactobacillus, have been of interest as treatment options, however, little is known about the molecular mechanisms of Lactobacillus-mediated inhibition of H. pylori pathogenesis. In this work, we investigated the effect of Lactobacillus culture supernatants, so-called conditioned medium (CM), from two gastric isolates, L. gasseri and L. oris, on the expression of transcriptional regulators in H. pylori. Among the four known two-component systems (TCSs), i.e., ArsRS, FlgRS, CheAY and CrdRS, the flagellar regulator gene flgR and the acid resistance associated arsS gene were down-regulated by L. gasseri CM, whereas expression of the other TCS-genes remained unaffected. L. gasseri CM also reduced the motility of H. pylori, which is in line with reduced flgR expression. Furthermore, among six transcription factors of H. pylori only the ferric uptake regulator gene fur was regulated by L. gasseri CM. Deletion of fur further led to dramatically increased sensitivity to the antimicrobial peptide LL-37. Taken together, the results highlight that released/secreted factors of some lactobacilli, but not all, downregulate transcriptional regulators involved in motility, acid tolerance and LL-37 sensitivity of H. pylori.

Lactate-Induced Dispersal of Neisseria meningitidis Microcolonies Is Mediated by Changes in Cell Density and Pilus Retraction and Is Influenced by Temperature Change.

Neisseria meningitidis is the etiologic agent of meningococcal meningitis and sepsis. Initial colonization of meningococci in the upper respiratory tract epithelium is crucial for disease development. The colonization occurs in several steps and expression of type IV pili (Tfp) is essential for both attachment and microcolony formation of encapsulated bacteria. Previously, we have shown that host-derived lactate induces synchronized dispersal of meningococcal microcolonies. In this study, we demonstrated that lactate-induced dispersal is dependent on bacterial concentration but not on the quorum-sensing system autoinducer-2 or the two-component systems NarP/NarQ, PilR/PilS, NtrY/NtrX, and MisR/MisS. Further, there were no changes in expression of genes related to assembly, elongation, retraction, and modification of Tfp throughout the time course of lactate induction. By using pilT and pptB mutants, however, we found that lactate-induced dispersal was dependent on PilT retraction but not on phosphoglycerol modification of Tfp even though the PptB activity was important for preventing reaggregation postdispersal. Furthermore, protein synthesis was required for lactate-induced dispersal. Finally, we found that at a lower temperature, lactate-induced dispersal was delayed and unsynchronized, and bacteria reformed microcolonies. We conclude that lactate-induced microcolony dispersal is dependent on bacterial concentration, PilT-dependent Tfp retraction, and protein synthesis and is influenced by environmental temperature.

Putative Adhesion Factors in Vaginal Lactobacillus gasseri DSM 14869: Functional Characterization.

Lactobacilli play an important role in the maintenance of a healthy vaginal microbiota, and some select species are widely used as probiotics. Vaginal isolates of Lactobacillus gasseri DSM 14869 and Lactobacillus rhamnosus DSM 14870 were previously selected to develop the probiotic EcoVag capsules and showed therapeutic effects in women with bacterial vaginosis (BV). However, the molecular mechanisms involved in their probiotic activity are largely unknown. In this study, we identified three cell surface molecules in L. gasseri DSM 14869 that promote adhesion to vaginal epithelial cells (VEC) by constructing dedicated knockout mutants, including exopolysaccharides (EPSs), a protein containing MucBP-like domains (N506_1778), and a putative novel adhesin (N506_1709) with rib/alpha-like domain repeats. EPS knockout mutants revealed 20-fold and 14-fold increases in adhesion to Caco-2 and HeLa cells, respectively, compared with wild type, while the adhesion to VEC was reduced 30% by the mutation, suggesting that EPSs might mediate tissue tropism for vaginal cells. A significant decrease in adhesion to Caco-2 cells, HeLa cells, and VEC was observed in the N506_1778 knockout mutant. The N506_1709 mutant showed no significant difference for the adhesion to Caco-2 and HeLa cells compared with wild type (WT); in contrast, the adhesion to VEC revealed a significant decrease (42%), suggesting that N506_1709 might mediate specific binding to stratified squamous epithelial cells, and this putative novel adhesin was annotated as Lactobacillus vaginal epithelium adhesin (LVEA). Thus, we have discovered an important role for EPSs and a novel adhesin, LVEA, in the adhesive capacity of a vaginal probiotic Lactobacillus strain.IMPORTANCE Lactobacilli are known to contribute to the maintenance of a healthy vaginal microbiota and some are selected as probiotics for the prevention or treatment of urogenital diseases, such as bacterial vaginosis. However, the molecular mechanisms for these health-promoting effects are not fully understood. Here, we functionally identified three cell surface factors of a Lactobacillus gasseri strain potentially involved in its adhesion to vaginal epithelial cells, including exopolysaccharides (EPSs) and two sortase-dependent proteins (N506_1778 and N506_1709). We could demonstrate the tissue-specific adhesion of EPSs to vaginal cells and that N506_1709 might be a novel adhesin specifically mediating bacterial binding to stratified squamous epithelial cells. The results provide important new information on the molecular mechanisms of vaginal Lactobacillus spp. adhesion.

An exploratory pilot study evaluating the supplementation of standard antibiotic therapy with probiotic lactobacilli in south African women with bacterial vaginosis.

BACKGROUND: To reduce acquisition and relapse of bacterial vaginosis (BV), lactobacilli must be maintained in the vaginal microbiome. Probiotic lactobacilli may aid this purpose. We investigated whether vaginal probiotics (containing Lactobacillus rhamnosus DSM 14870 and Lactobacillus gasseri DSM 14869) would result in vaginal colonisation with lactobacilli in women with and without BV. METHODS: This prospective, partially randomised, exploratory pilot study was conducted in Soweto, South Africa. Thirty-nine sexually-active, HIV negative women were enrolled from October 2014 to May 2016 into three arms. Women who did not have BV (Group 1, n = 13) self-administered probiotic capsules vaginally once daily for 30 days, then once a week until Day 190. Women diagnosed with BV were randomized into Group 2 (n = 12) or Group 3 (n = 14) and treated with the triple oral antibiotic combination for vaginal discharge syndrome per South African guidelines (cefixime 400 mg stat, doxycycline 100 mg BD for 7 days and metronidazole 2 g stat). Immediately after antibiotic treatment, women in Group 2 self-administered probiotic capsules vaginally once daily for 30 days then vaginally once a week until Day 190. Women in Group 3 were not given lactobacilli. RESULTS: During the study, L. rhamnosus DSM 14870 or L. gasseri DSM 14869, were isolated in 5/13 (38.5%) women in Group 1 compared to 10/12 (83.3%) women in Group 2 (p = 0.041). The 1-month and 6-month BV cure rates were similar (P >  0.05) between Group 2 (42 and 25%) compared to Group 3 (36 and 25%). In Group 2, no correlation was observed between the frequency of isolation of the two Lactobacillus strains and the 1-month or 6-month cure rate. CONCLUSIONS: Supplementation with vaginal probiotic capsules resulted in colonisation of the vagina by the Lactobacillus strains (L. rhamnosus DSM 14870 and L. gasseri DSM 14869) contained in the capsules. We observed low initial cure rates of BV after a stat dose of metronidazole and that the probiotic did not improve BV cure rates or alleviate recurrence which could be due to treatment failure or very limited power of the study. TRIAL REGISTRATION: Registered at the Pan African Clinical Trial Registry ( www.pactr.org ) on April 13, 2018 (retrospectively registered). Trial identification number: PACTR201804003327269.

Recombinant Lactococcus lactis expressing bioactive exendin-4 to promote insulin secretion and beta-cell proliferation in vitro.

In recent years, therapeutic peptides have garnered great interest in the pharmaceutical industry for the treatment of diabetes. Lactic acid bacteria (LAB) are an appealing vehicle for safe and convenient oral delivery of bioactive peptide and protein drugs. Exendin-4 (Exd4) is a glucagon-like protein-1 (GLP-1) receptor agonist that is considered an excellent therapeutic peptide drug for type 2 diabetes due to its longer-lasting bioactivity, resulting from resistance to dipeptidyl peptidase 4. We explored Lactococcus lactis with the nisin-controlled gene expression (NICE) system as an oral delivery system for recombinant (r) Exd4 peptide in situ. Heterologous expression and secretion of rExd4 by L. lactis NZ9000/pNZ8048-rExd4 were successful and efficient under the NICE system. In vitro treatment with rExd4 significantly enhanced insulin secretion of INS-1 cells and activated the PI3-K/AKT signal pathway with protein levels of AKT and p-AKT increasing 1.6- to 1.8-fold compared to negative controls, similar to the positive GLP-1 controls. INS-1 cells treated with rExd4 also showed enhanced proliferation and inhibited apoptosis, corresponding with the effects of the standard Exd4 and GLP-1 treatments. Our data suggest that the rExd4 secreted by L. lactis is a bioactive insulinotropic peptide and functional GLP-1 receptor agonist that enhances glucose-dependent insulin secretion and activates the PI3-K/AKT signal pathway; furthermore, it may be involved in improving proliferation and inhibiting apoptosis of INS-1 cells in in vitro treatments. Therefore, L. lactis producing rExd4 may potentially serve as a novel strategy for oral treatment of diabetes.

Recombinant Lactococcus lactis NZ9000 secretes a bioactive kisspeptin that inhibits proliferation and migration of human colon carcinoma HT-29 cells.

BACKGROUND: Proteinaceous bioactive substances and pharmaceuticals are most conveniently administered orally. However, the facing problems are the side effects of proteolytic degradation and denaturation in the gastrointestinal tract. In recent years, lactic acid bacteria (LAB) have been verified to be a promising delivery vector for susceptible functional proteins and drugs. KiSS-1 peptide, a cancer suppressor, plays a critical role in inhibiting cancer metastasis and its activity has been confirmed by direct administration. However, whether this peptide can be functionally expressed in LAB and exert activity on cancer cells, thus providing a potential alternative administration manner in the future, has not been demonstrated. RESULTS: A recombinant Lactococcus lactis strain NZ9000-401-kiss1 harboring a plasmid containing the gene of the tumor metastasis-inhibiting peptide KiSS1 was constructed. After optimization of the nisin induction conditions, the recombinant strain efficiently secreted KiSS1 with a maximum detectable amount of 27.9 µg/ml in Dulbecco's Modified Eagle medium. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide and would healing assays, respectively, indicated that the secreted KiSS1 peptide remarkably inhibited HT-29 cell proliferation and migration. Furthermore, the expressed KiSS1 was shown to induce HT-29 cell morphological changes, apoptosis and reduce the expression of matrix metalloproteinase 9 (MMP-9) at both mRNA and protein levels. CONCLUSIONS: A recombinant L. lactis NZ9000-401-kiss1 successfully expressing the human kiss1 was constructed. The secreted KiSS1 peptide inhibited human HT-29 cells' proliferation and migration probably by invoking, or mediating the cell-apoptosis pathway and by down regulating MMP-9 expression, respectively. Our results suggest that L. lactis is an ideal cell factory for secretory expression of tumor metastasis-inhibiting peptide KiSS1, and the KiSS1-producing L. lactis strain may serve as a new tool for cancer therapy in the future.

Overexpression of Small Heat Shock Protein Enhances Heat- and Salt-Stress Tolerance of Bifidobacterium longum NCC2705.

Bifidobacteria are probiotics that are incorporated live into various dairy products. They confer health-promotive effects via gastrointestinal tract colonization. However, to provide their health-beneficial properties, they must battle the various abiotic stresses in that environment, such as bile salts, acids, oxygen, and heat. In this study, Bifidobacterium longum salt- and heat-stress tolerance was enhanced by homologous overexpression of a small heat shock protein (sHsp). A positive contribution of overproduced sHsp to abiotic stress tolerance was observed when the bacterium was exposed to heat and salt stresses. Significantly higher survival of B. l ongum NCC2705 overexpressing sHsp was observed at 30 and 60 min into heat (55 °C) and salt (5 M NaCl) treatment, respectively. Thermotolerance analysis at 47 °C with sampling every 2 h also revealed the great potential tolerance of the engineered strain. Cell density and acid production rate increased for the sHsp-overexpressing strain after 8 and 10 h of both heat and salt stresses. In addition, tolerance to bile salts, low pH (3.5) and low temperature (4 °C) was also increased by homologous overexpression of the sHsp hsp20 in B. l ongum. Results revealed that hsp20 overexpression in B longum NCC2705 plays a positive cross-protective role in upregulating abiotic responses, ensuring the organism's tolerance to various stress conditions; therefore, sHsp-overexpressing B. l ongum is advised for fermented dairy foods and other probiotic product applications.

Combination of heterogeneous catalase and superoxide dismutase protects Bifidobacterium longum strain NCC2705 from oxidative stress.

Bifidobacteria are generally sensitive to oxidative stress caused by reactive oxygen species (ROS). To improve oxidative-stress tolerance, the superoxide dismutase (SOD) gene from Streptococcus thermophilus (StSodA) and the heme-dependent catalase (KAT) gene from Lactobacillus plantarum (LpKatL) were heterologously expressed in Bifidobacterium longum strain NCC2705. Three types of strain NCC2705 transformants were obtained: with transgenic SOD expression, with transgenic KAT expression, and with coexpression of the two genes. Intracellular expression of the genes and their functional role in oxidative-stress resistance were evaluated. In response to oxidative stress, B. longum NCC2705/pDP401-LpKatL (expressing LpKatL) and NCC2705/pDP-Kat-Sod (coexpressing LpKatL and StSodA) rapidly degraded exogenous H2O2 and the peroxides generated as a byproduct of aerobic cultivation, preventing oxidative damage to DNA and RNA. Individual expression of StSodA or LpKatL both improved B. longum NCC2705 cell viability. Survival rate of strain NCC2705 was further improved by combining SOD and KAT expression. The two enzymes played complementary roles in ROS-scavenging pathways, and coexpression led to a synergistic beneficial effect under conditions of intensified oxidative stress. Our results illustrate that heterogeneous expression of heme-dependent KAT and Mn(2+)-dependent SOD is functional in the B. longum oxidative-stress response, and synergistic protection is achieved when their expressions are combined.

Design, structure and plasma binding of ancestral ß-CoV scaffold antigens.

We report the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interact with plasma of patients recovered from COVID-19 but do not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yield high resolution structures (2.6-2.8 Å) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. The structures reveal an intricate hydrogen-bonding network mediated by well-resolved loops, both within and across monomers, tethering the N-terminal domain and RBD together. We show that AnSA-5 can induce and boost a broad-spectrum immune response against the wild-type RBD as well as circulating variants of concern in an immune organoid model derived from tonsils. Finally, we highlight how AnSAs are potent scaffolds by replacing the ancestral RBD with the wild-type sequence, which restores ACE2 binding and increases the interaction with convalescent plasma.

19n01, a broadly neutralizing antibody against omicron BA.1, BA.2, BA.4/5, and other SARS-CoV-2 variants of concern.

This study reports the isolation and characterization of a human monoclonal antibody (mAb) called 19n01. This mAb was isolated by using single-cell RNAseq of B cells from donors infected with the ancestral strain. This mAb possesses a potent and broad capacity to bind and neutralize all previously circulating variants of concern (VOCs), including Omicron sublineages BA.1, BA.2, and BA.4/5. The pseudovirus neutralization assay revealed robust neutralization capacity against the G614 strain, BA.1, BA.2, and BA.4/5, with inhibitory concentration (IC50) values ranging from 0.0035 to 0.0164 µg/mL. The microneutralization assay using the G614 strain and VOCs demonstrated IC50 values of 0.013-0.267 µg/mL. Biophysical and structural analysis showed that 19n01 cross-competes with ACE2 binding to the receptor-binding domain (RBD) and the kinetic parameters confirmed the high affinity against the Omicron sublineages (KD of 61 and 30 nM for BA.2 and BA.4/5, respectively). These results suggest that the 19n01 is a remarkably potent and broadly reactive mAb.

Lactate causes downregulation of Helicobacter pylori adhesin genes sabA and labA while dampening the production of proinflammatory cytokines.

Chronic inflammation induced by Helicobacter pylori is strongly associated with gastric cancer development, which is influenced by both bacterial virulence and host genetics. The sialic acid-binding adhesin SabA and the MUC5AC-binding adhesin LabA are important H. pylori virulence factors that facilitate adhesion of the bacterium, which is a crucial step in colonization. Lactate utilization has been reported to play a key role in the pathogenicity of different bacterial species. However, this is poorly understood in H. pylori. In this study, we investigated the effect of lactate on H. pylori adhesin gene expression and the regulation of host inflammatory cytokines. We show that the bacterial adhesins SabA and LabA were downregulated at the transcriptional level during incubation of H. pylori with lactate. Downregulation of sabA required the involvement of the two-component system ArsRS, while labA was regulated via the CheA/CheY system, indicating differences in the regulation of these genes in response to lactate. The levels of the proinflammatory cytokines TNF and IL-6 in H. pylori-stimulated macrophages were reduced when lactate was present. Interestingly, glucose did not prevent the secretion of these cytokines. Taken together, our data suggest that lactate affects H. pylori adhesin gene expression and the host response upon infection.

Lactobacilli as a Vector for Delivery of Nanobodies against Norovirus Infection.

Passive administration of neutralizing antibodies (Abs) is an attractive strategy for the control of gastrointestinal infections. However, an unanswered practical concern is the need to assure the stability of sufficient amounts of orally administered neutralizing Abs against intestinal pathogens (e.g., norovirus) in the harsh environment of the gastrointestinal tract. To this end, we expressed a single-domain Ab (VHH, nanobody) against norovirus on the cell surface of Lactobacillus, a natural and beneficial commensal component of the gut microbiome. First, we used intestinal epithelial cells generated from human induced pluripotent stem cells to confirm that VHH 1E4 showed neutralizing activity against GII.17 norovirus. We then expressed VHH 1E4 as a cell-wall-anchored form in Lactobacillus paracasei BL23. Flow cytometry confirmed the expression of VHH 1E4 on the surface of lactobacilli, and L. paracasei that expressed VHH 1E4 inhibited the replication of GII.17 norovirus in vitro. We then orally administered VHH 1E4-expressing L. paracasei BL23 to germ-free BALB/c mice and confirmed the presence of lactobacilli with neutralizing activity in the intestine for at least 10 days after administration. Thus, cell-wall-anchored VHH-displaying lactobacilli are attractive oral nanobody deliver vectors for passive immunization against norovirus infection.

Lung function before and after COVID-19 in young adults: A population-based study.

Background: There is limited evidence on the long-term impact of mild-to-moderate coronavirus disease 2019 (COVID-19) on lung function among young adults. Objectives: We aimed to assess whether COVID-19 has a negative impact on lung function in young adults and whether asthma, allergic sensitization, or use of inhaled corticosteroids (ICSs) modifies a potential association. Methods: Participants from the population-based BAMSE (Barn, Allergi, Miljö, Stockholm, Epidemiologi) cohort with spirometry assessed before (2016-2019) and after onset of the COVID-19 pandemic (2020-2021) were included. Serum levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain-specific IgG, IgM, and/or IgA (determined with ELISA) defined seropositivity. Mean change in lung function (ie, change in FEV1, forced vital capacity [FVC], and FEV1/FVC ratio expressed as percent of predicted [pp]) from before to after onset of the pandemic were compared between the seronegative and seropositive participants. In seropositive participants, change in lung function was assessed in relation to allergic sensitization and self-reported ICS use. Results: Of the 853 included participants, 29% (n = 243) were seropositive. There were no differences in change in lung function between the seronegative and seropositive participants (for mean change in FEV1 pp [SD], seropositivity = 0.87% [4.79%] and seronegativity = 1.03% (4.76%) [P = .66] for difference using a t test; FVC pp (SD), seropositivity = 1.34% (4.44%) and seronegativity = 1.29% (4.27%) [P = .87]; and for FEV1/FVC pp (SD), seropositivity = -0.25% (3.13%) and seronegativity = -0.13% (3.15%) [P = .61]). Similar results were observed among participants with asthma (n = 147 [17%]). Among seropositive participants, allergic sensitization or ICS use did not influence lung function. Conclusion: We found no evidence of mild-to-moderate COVID-19 affecting lung function long term in a population-based cohort of young adults. Moreover, neither asthma nor allergic sensitization nor ICS use affected the results.