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.

Comparative single-cell transcriptomic profile of hybrid immunity induced by adenovirus vector-based COVID-19 vaccines.

In this study, antibody response and a single-cell RNA-seq analysis were conducted on peripheral blood mononuclear cells from five different groups: naïve subjects vaccinated with AZD1222 (AZ) or Ad5-nCoV (Cso), individuals previously infected and later vaccinated (hybrid) with AZD1222 (AZ-hb) or Ad5-nCoV (Cso-hb), and those who were infected and had recovered from COVID-19 (Inf). The results showed that AZ induced more robust neutralizing antibody responses than Cso. The single-cell RNA data revealed a high frequency of memory B cells in the Cso and Cso-hb. In contrast, AZ and AZ-hb groups exhibited the highest proportion of activated naïve B cells expressing CXCR4. Transcriptomic analysis of CD4+ and CD8+ T cells demonstrated a heterogeneous response following vaccination, hybrid immunity, or natural infection. However, a single dose of Ad5-nCoV was sufficient to strongly activate CD4+ T cells (naïve and memory) expressing ANX1 and FOS, similar to the hybrid response observed with AZ. An interesting finding was the robust activation of a subset of CD8+ T cells expressing GZMB, GZMH, and IFNG genes in the Cso-hb group. Our findings suggest that both vaccines effectively stimulated the cellular immune response; however, the Ad5-nCoV induced a more robust CD8+ T-cell response in previously infected individuals.

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.

A Heterodimeric Antibody Fragment for Passive Immunotherapy Against Norovirus Infection.

Human noroviruses cause an estimated 685 million infections and 200 000 deaths annually worldwide. Although vaccines against GII.4 and GI.1 genotypes are under development, no information is available regarding vaccines or monoclonal antibodies to other noroviral genotypes. Here, we developed 2 variable-domain llama heavy-chain antibody fragment (VHHs) clones, 7C6 and 1E4, against GII.4 and GII.17 human noroviruses, respectively. Although 7C6 cross-reacted with virus-like particles (VLPs) of GII.17, GII.6, GII.3, and GII.4, it neutralized only GII.4 norovirus. In contrast, 1E4 reacted with and neutralized only GII.17 VLPs. Both VHHs blocked VLP binding to human induced pluripotent stem cell-derived intestinal epithelial cells and carbohydrate attachment factors. Using these 2 VHHs, we produced a heterodimeric VHH fragment that neutralized both GII.4 and GII.17 noroviruses. Because VHH fragments are heat- and acid-stable recombinant monoclonal antibodies, the heterodimer likely will be useful for oral immunotherapy and prophylaxis against GII.4 and GII.17 noroviruses in young, elderly, or immunocompromised persons.

Clinical implications of experimental analyses of AID function on predictive computational tools: Challenge of missense variants.

Due to the increased usage of high throughput sequencing for the diagnosis of genetically inherited disorders, it is vital to evaluate the risk of new variants and novel genes before accepting them in clinical practice. However, discordant in silico and in vitro results, challenge estimations of the effect of an identified genetic variant. We aimed to comprehensively evaluate pathogenic and polymorphic variants using the activation-induced-cytidine-deaminase (AICDA) gene as a model. We systematically searched and identified patients with confirmed AICDA-mutations. Population-based-databases were screened for germline-polymorphic-AICDA-variants. Activity of AICDA-mutant and severity of the clinical and immunologic-phenotype were showed comparing 108 population-based-variants with 48 pathogenic mutations (12 overlapping-variants). Discordant predictions of different algorithms were observed on average in 38% of the population-database variants, mainly for missense mutations. Functional activity in mutations observed only in patients was significantly lower than variants in the population databases and overlapping-variants between patients and the general-population. Surprisingly, overlapping-variants had an even higher functional activity than the most common polymorphic-variants; however, their pathogenicity was still distinguishable when their function was compared with wild-type AICDA. Classifications of genetic variants cannot readily be translated into a clinical implication. Combined databases of functional and computational assays should therefore be developed for each specific gene.

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.

Clostridium difficile, the Difficult "Kloster" Fuelled by Antibiotics.

Clostridium difficile is normally present in low numbers in a healthy adult gastro-intestinal tract (GIT). Drastic changes in the microbial population, e.g., dysbiosis caused by extensive treatment with antibiotics, stimulates the growth of resistant strains and the onset of C. difficile infection (CDI). Symptoms of infection varies from mild diarrhea to colitis (associated with dehydration and bleeding), pseudomembranous colitis with yellow ulcerations in the mucosa of the colon, to fulminant colitis (perforation of the gut membrane), and multiple organ failure. Inflamed epithelial cells and damaged mucosal tissue predisposes the colon to other opportunistic pathogens such as Clostridium perfringens, Staphylococcus aureus, Klebsiella oxytoca, Candida spp., and Salmonella spp. This may lead to small intestinal bacterial overgrowth (SIBO), sepsis, toxic megacolon, and even colorectal cancer. Many stains of C. difficile are resistant to metronidazole and vancomycin. Vaccination may be an answer to CDI, but requires more research. Success in treatment with probiotics depends on the strains used. Oral or rectal fecal transplants are partly effective, as spores in the small intestine may germinate and colonize the colon. The effect of antibiotics on C. difficile and commensal gut microbiota is summarized and changes in gut physiology are discussed. The need to search for non-antibiotic methods in the treatment of CDI and C. difficile-associated disease (CDAD) is emphasized.

Lactobacillus delivery of bioactive interleukin-22.

BACKGROUND: Interleukin-22 (IL-22) plays a prominent role in epithelial regeneration and dampening of chronic inflammatory responses by protecting intestinal stem cells from immune-mediated tissue damage. IL-22 has a considerable therapeutic potential in graft-versus-host disease (GVHD), which is a frequent and challenging complication following allogeneic stem cell transplantation. The aim of our study was to engineer Lactobacillus for delivery of IL-22 directly to the intestinal mucosa as a new therapeutic strategy for GVHD. RESULTS: The secretion and surface anchoring of mouse IL-22 by Lactobacillus paracasei BL23 was demonstrated by Western blot and flow cytometry. Both secreted and anchored mouse IL-22 produced by Lactobacillus was biologically active, as determined by its ability to induce IL-10 secretion in the Colo 205 human colon cancer cell line. CONCLUSIONS: We have demonstrated the secretion and surface anchoring of bioactive IL-22 by Lactobacillus. Our results suggest that IL-22 expressing lactobacilli may potentially be a useful mucosal therapeutic agent for the treatment of GVHD, provided that chromosomal integration of the IL-22 expression cassettes can be achieved.

Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies.

Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA(-) TcdB(+) strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients.

An Exopolysaccharide-Deficient Mutant of Lactobacillus rhamnosus GG Efficiently Displays a Protective Llama Antibody Fragment against Rotavirus on Its Surface.

Rotavirus is the leading cause of infantile diarrhea in developing countries, where it causes a high number of deaths among infants. Two vaccines are available, being highly effective in developed countries although markedly less efficient in developing countries. As a complementary treatment to the vaccines, a Lactobacillus strain producing an anti-rotavirus antibody fragment in the gastrointestinal tract could potentially be used. In order to develop such an alternative therapy, the effectiveness of Lactobacillus rhamnosus GG to produce and display a VHH antibody fragment (referred to as anti-rotavirus protein 1 [ARP1]) on the surface was investigated. L. rhamnosus GG is one of the best-characterized probiotic bacteria and has intrinsic antirotavirus activity. Among four L. rhamnosus GG strains [GG (CMC), GG (ATCC 53103), GG (NCC 3003), and GG (UT)] originating from different sources, only GG (UT) was able to display ARP1 on the bacterial surface. The genomic analysis of strain GG (UT) showed that the genes welE and welF of the EPS cluster are inactivated, which causes a defect in exopolysaccharide (EPS) production, allowing efficient display of ARP1 on its surface. Finally, GG (UT) seemed to confer a level of protection against rotavirus-induced diarrhea similar to that of wild-type GG (NCC 3003) in a mouse pup model, indicating that the EPS may not be involved in the intrinsic antirotavirus activity. Most important, GG (EM233), a derivative of GG (UT) producing ARP1, was significantly more protective than the control strain L. casei BL23.

Vaginal colonisation by probiotic lactobacilli and clinical outcome in women conventionally treated for bacterial vaginosis and yeast infection.

BACKGROUND: The aim of this study was to investigate the colonisation by lactobacilli and clinical outcome in women with bacterial vaginosis (BV) and recurrent vulvovaginal candidiasis (R-VVC) receiving antibiotic or anti-fungal treatment in combination with the probiotic EcoVag® capsules. METHODS: A total of 40 Scandinavian women diagnosed with BV or VVC on the basis of Amsel's criteria or clinical symptoms were consecutively recruited in two pilot open label clinical trials. In trial I, women with BV were treated with clindamycin and metronidazole followed by vaginal EcoVag® capsules, containing Lactobacillus rhamnosus DSM 14870 and Lactobacillus gasseri DSM 14869, for 5 consecutive days after each antibiotic treatment. In trial II, women were recruited in three groups as follows: women with BV receiving clindamycin and metronidazole treatment together with a prolonged administration of EcoVag® (10 consecutive days after each antibiotic treatment followed by weekly administration of capsules for next four months), women with R-VVC receiving extended fluconazole and EcoVag® treatment, and women receiving extended fluconazole treatments only. The difference in frequency of isolation of EcoVag® strains or other lactobacilli between groups was compared by Fisher's exact test. RESULTS: The 6-month cure rate for BV was 50 % in trial I while both the 6- and 12-month cure rates were 67 % in trial II. The 6- and 12-month cure rates for VVC were 100 % and 89 % in women receiving fluconazole and EcoVag®, and 100 % and 70 % in women receiving fluconazole only. The frequency of isolation of any Lactobacillus species during the course of the study was associated with cure of BV in trial I and II, whereas the frequency of isolation of EcoVag® strains was significantly associated with the cure of BV in trial II only. As previously observed, a change in sexual partner was associated with relapse of BV with an Odds ratio of 77 (95 % CI: 2.665 to 2225). CONCLUSIONS: The study suggests that the treatment with antibiotics or anti-fungal medication in combination with EcoVag® capsules provide long-term cure against BV and R-VVC as compared to previous reports. TRIAL REGISTRATION: ClinicalTrials.gov NCT02295579 . Registered November 20, 2014.

Identification and characterisation of vaginal lactobacilli from South African women.

BACKGROUND: Bacterial vaginosis (BV), which is highly prevalent in the African population, is one of the most common vaginal syndromes affecting women in their reproductive age placing them at increased risk for sexually transmitted diseases including infection by human immunodeficiency virus-1. The vaginal microbiota of a healthy woman is often dominated by the species belonging to the genus Lactobacillus namely L. crispatus, L. gasseri, L. jensenii and L. iners, which have been extensively studied in European populations, albeit less so in South African women. In this study, we have therefore identified the vaginal Lactobacillus species in a group of 40 African women from Soweto, a township on the outskirts of Johannesburg, South Africa. METHODS: Identification was done by cultivating the lactobacilli on Rogosa agar, de Man-Rogosa-Sharpe (MRS) and Blood agar plates with 5% horse blood followed by sequencing of the 16S ribosomal DNA. BV was diagnosed on the basis of Nugent scores. Since some of the previous studies have shown that the lack of vaginal hydrogen peroxide (H2O2) producing lactobacilli is associated with bacterial vaginosis, the Lactobacillus isolates were also characterised for their production of H2O2. RESULTS: Cultivable Lactobacillus species were identified in 19 out of 21 women without BV, in three out of five women with intermediate microbiota and in eight out of 14 women with BV. We observed that L. crispatus, L. iners, L. jensenii, L. gasseri and L. vaginalis were the predominant species. The presence of L. crispatus was associated with normal vaginal microbiota (P = 0.024). High level of H2O2 producing lactobacilli were more often isolated from women with normal microbiota than from the women with BV, although not to a statistically significant degree (P = 0.064). CONCLUSION: The vaginal Lactobacillus species isolated from the cohort of South African women are similar to those identified in European populations. In accordance with the other published studies, L. crispatus is related to a normal vaginal microbiota. Hydrogen peroxide production was not significantly associated to the BV status which could be attributed to the limited number of samples or to other antimicrobial factors that might be involved.

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.