Safety and immunogenicity of a single dose of Tdap compared to Td in pregnant women in Mali and 3 its effect on infant immune responses: a single-centre, randomised, double-blind, active-controlled phase 2 study.

Background: While maternal pertussis vaccination is a strategy to reduce infant morbidity, safety and immunogenicity data are limited in sub-Saharan Africa. We aimed to evaluate the safety of a single dose of tetanus, diphtheria and acellular pertussis vaccine (Tdap) vaccine compared to tetanus and diphtheria vaccine (Td) vaccine in pregnant women in Bamako, Mali and to assess the pertussis toxin (PT) antibody response at birth. Methods: In this phase 2, single-centre, randomised, double-blind, active-controlled study, from 23 January 2019 to 10 July 2019, healthy 18-39 year old women in the second trimester of a singleton pregnancy were randomised 2:1 to receive Tdap or Td. Blood was tested for serum immunoglobulin G (IgG) against PT and other vaccine antigens using a qualified Meso Scale Discovery multiplex immunoassay. The co-primary objectives evaluated safety and birth anti-PT levels. Infant immune responses to whole-cell pertussis vaccine (DTwP) were assessed. Statistical analysis was descriptive. This trial is registered with clinicaltrials.gov, NCT03589768. Findings: 133 women received Tdap and 67 received Td, with 126 and 66 livebirths, respectively. In the Tdap group, 22 serious adverse events (SAEs) including one maternal death occurred in 20 participants (15·0%), with 10 SAEs in 10 participants (14·9%) in the Td group. Among infants, 18 events occurred among 13 participants (10.3%) and 8 SAEs in 6 participants (9.1%), including three and two infant deaths, occurred in Tdap and Td groups, respectively. None were related to study vaccines. Anti-PT geometric mean concentration (GMC) at birth in the Tdap group was higher than in the Td group (55.4 [46.2-66.6] IU/ml vs 7.9 [5.4-11.5] IU/ml). One month after the third dose of DTwP, the GMC in infants born to mothers in the Tdap group were lower compared to the Td group (20.2 [13.7-29.9] IU/ml vs 77.2 [32.2-184.8] IU/ml). By 6 months of age, the anti- PT GMCs were 17.3 [12.8-23.4] IU/ml and 67.1 [35.5-126.7] IU/ml in Tdap and Td groups, respectively. At birth, anti-tetanus toxin (TT) GMCs were higher in infants in the Td vs Tdap group (5.9 [5.0-7.0] IU/ml vs 4.1 [3.5-4.8] IU/ml). Anti-diphtheria toxin GMCs were similar in both groups. Interpretation: Tdap administered to pregnant women in Mali is safe and well-tolerated. Infants of mothers who received Tdap were born with high PT and protective anti-TT antibody levels. By six months of age, after primary vaccination, the PT levels were lower in the Tdap group compared to the Td group. The blunted immune responses to primary DTwP vaccination in the Tdap infant group warrant further study. Funding: This project was funded by National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), under contract numbers 75N93021C00012 (The Emmes Company), and HHSN27220130000221 (University of Maryland, Baltimore). Dr. Susana Portillo was supported by NIH award no. T32AI007524. NIAID, NIH provided Tdap vaccine (BOOSTRIX).

Quantitative analysis of pertussis, tetanus, and diphtheria antibodies in sera and breast milk from Tdap vaccinated women using a qualified multiplex assay.

Pertussis (whooping cough) is a reemergent, highly contagious respiratory infection of public health concern. Infants prior to initiation of their primary vaccination series are the most vulnerable to severe infection, and even death. Vaccination during pregnancy is an efficacious means of reducing infection in infants. This approach relies on boosting maternal immunity and passive transfer of antibodies to the infant via placenta and breast milk. Similarly, maternal vaccination post-partum can enhance maternal-infant immunity. To support the analysis of pertussis immunity in the context of maternal-infant immunization, we developed a high throughput multiplex assay for simultaneous quantification of serum IgG antibodies against pertussis vaccine antigens: pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (FIM2/3), and against tetanus (TT) and diphtheria toxoids (DT), using the Meso Scale Discovery (MSD) platform. The assay was qualified, and specificity, sensitivity, accuracy, precision, linearity, and robustness were demonstrated. The assay was subsequently adapted for quantification of IgG and IgA in breast milk. Applied to a serological survey of pregnant women living in the United States and sub-Saharan Africa, this method revealed differences in magnitude and breadth of antibody profile, consistent with history of vaccination. A longitudinal analysis of Tdap responses in women vaccinated post-partum demonstrated a rapid increase in serum IgG that remained elevated for up to 24 months. Likewise, high levels of vaccine-specific IgA and IgG antibodies were present in breast milk, although they exhibited faster decay. This multiplex MSD assay is a reliable and practical tool for quantification of pertussis, tetanus, and diphtheria antibodies in serum and breast milk in serosurveys or vaccine studies. IMPORTANCE: Pertussis (whooping cough) has reemerged in recent years. Vaccination during pregnancy is an effective approach to prevent illness during the first months of life. We developed a multiplex assay for quantification of pertussis, tetanus, and diphtheria serum antibodies using the Meso Scale Discovery (MSD) platform; the method was qualified, and specificity, precision, accuracy, linearity, and limits of quantification were defined. It was also adapted for quantification of antibodies in breast milk. We successfully determined serostatus in women from different regions and with different vaccination histories, as well as responses to Tdap in blood and breast milk post-partum. This is the first description of a multiplex assay for the quantification of pertussis, tetanus, and diphtheria antibodies in breast milk.

Recombinant full-length Plasmodium falciparum circumsporozoite protein-based vaccine adjuvanted with GLA-LSQ: Results of Phase 1 testing with malaria challenge.

INTRODUCTION: Malaria is preventable yet causes >600,000 deaths annually. RTS, S, the first marketed malaria vaccine, has modest efficacy, but improvements are needed for eradication. METHODS: We conducted an open-label, dose escalation Phase 1 study of a recombinant, full-length circumsporozoite protein vaccine (rCSP) administered with adjuvant GLA-LSQ on days 1, 29, and 85 or 1 and 490 to healthy, malaria-naïve adults. Primary endpoints were safety and reactogenicity. Secondary endpoints were antibody responses and Plasmodium falciparum parasitemia after homologous controlled human malaria infection (CHMI). RESULTS: Participants were enrolled into four groups receiving rCSP/GLA-LSQ: 10 µg x 3 (n = 20), 30 µg x 3 (n = 10), 60 µg x 3 (n = 10) or 60 µg x 2 (n = 9); ten participants received 30 µg rCSP alone x 3; and six infectivity controls. Participants experienced no serious adverse events. Rates of solicited and unsolicited adverse events were similar among groups. All 26 participants who underwent CHMI 28 days after final vaccinations developed malaria. Increasing vaccine doses induced higher IgG titers, but did not achieve previously established RTS, S benchmarks. CONCLUSIONS: rCSP/GLA-LSQ had favorable safety results. However, tested regimens did not induce protective immunity. Further investigation could assess if adjuvant or schedule adjustments improve efficacy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03589794.

Shigella virulence protein VirG is a broadly protective antigen and vaccine candidate.

Diarrhea caused by Shigella has been associated with high morbidity and mortality in young children worldwide. There are no licensed vaccines, and those clinically advanced have restricted coverage as they elicit serotype-specific immunity while disease is caused by multiple circulating serotypes. Our group had previously reported a close association between serum antibodies to the Shigella virulence factor VirG (or IcsA) and clinical protection in infected individuals. VirG is highly conserved among Shigella strains and appealing as a broad-spectrum vaccine candidate. In this study, we investigated the immunogenicity and protective capacity of VirG as a subunit vaccine in mice. The surface-exposed alpha (α) domain of VirG (VirGα) was produced as a recombinant protein. This region has almost identical immune reactivity to full-length VirG. Administered intramuscularly with alum, VirGα elicited robust immune responses and high protective efficacy against S. flexneri 2a and S. sonnei. Almost complete protection was afforded by VirGα given intranasally with the E. coli double mutant heat-labile toxin (dmLT). VirGα-specific antibodies recognized VirG expressed on live Shigella, and blocked Shigella adhesion and invasion to human colonic cells. These results show for the first time that VirGα is a promising cross-protective vaccine candidate to prevent Shigella infection.

Oral immunization with Shigella sonnei WRSs2 and WRSs3 vaccine strains elicits systemic and mucosal antibodies with functional anti-microbial activity.

Shigella causes bacillary dysentery and is responsible for a high burden of disease globally. Several studies have emphasized the value of functional antibody activity to understand Shigella immunity and correlates of protection. The anti-microbial function of local (mucosal) antibodies and their contribution to preventing Shigella infection remain unknown. The goal of this study was to identify the functional humoral immune effectors elicited by two Shigella sonnei live oral vaccine candidates, WRSs2 and WRSs3. Complement-dependent bactericidal [serum bactericidal antibody (SBA)/bactericidal antibody (BA)] and opsonophagocytic killing antibody (OPKA) activity were determined in sera and stool extracts as indicators of systemic and local anti-microbial immunity. High levels of SBA/BA and OPKA were detected in serum as well as in fecal extracts from volunteers who received a single dose of WRSs2 and WRSs3. Functional antibody activity peaked on days 10 and 14 post-vaccination in fecal and serum samples, respectively. Bactericidal and OPKA titers were closely associated. Peak fold rises in functional antibody titers in serum and fecal extracts were also associated. Antibody activity interrogated in IgG and IgA purified from stool fractions identified IgG as the primary driver of mucosal bactericidal and OPKA activity, with minimal functional activity of IgA alone, highlighting an underappreciated role for IgG in bacterial clearance in the mucosa. The combination of IgG and IgA in equal proportions enhanced bactericidal and OPKA titers hinting at a co-operative or synergistic action. Our findings provide insight into the functional anti-microbial capacity of vaccine-induced mucosal IgG and IgA and propose an operative local humoral effector of protective immunity.IMPORTANCEThere is an urgent need for a safe, effective, and affordable vaccine against Shigella. Understanding the immunological underpinning of Shigella infection and the make-up of protective immunity is critical to achieve the best approach to prevent illness caused by this mucosal pathogen. We measured the complement-dependent bactericidal and opsonophagocytic antibody killing in serum and stool extracts from adult volunteers vaccinated with Shigella sonnei live oral vaccine candidates WRSs2 and WRSs3. For the first time, we detected functional antibody responses in stool samples that were correlated with those in sera. Using purified stool IgA and IgG fractions, we found that functional activity was mediated by IgG, with some help from IgA. These findings provide insight into the functional anti-microbial capacity of vaccine-induced mucosal IgG and IgA and support future studies to identify potential markers of protective mucosal immunity.

Detecting residual chronic Salmonella Typhi carriers on the road to typhoid elimination in Santiago, Chile, 2017-2019.

OBJECTIVES: In Santiago, Chile, where typhoid had been hyperendemic (1977-1991), we investigated whether residual chronic carriers could be detected among household contacts of non-travel-related typhoid cases occurring 2017-2019. METHODS: Culture-confirmed cases were classified as "autochthonous" (domestically-acquired) versus "travel/immigration-related". Household contacts of cases had stool cultures and serum Vi antibody measurements to detect chronic Salmonella Typhi carriers. Whole genome sequences of acute cases and their epidemiologically-linked chronic carrier isolates were compared. RESULTS: Five of 16 autochthonous typhoid cases (31.3%) were linked to four chronic carriers in case households; two cases (onsets 23 months apart) were linked to the same carrier. Carriers were women aged 69-79 years with gallbladder dysfunction and Typhi fecal excretion; three had highly elevated serum anti-Vi titers. Genomic analyses revealed close identity (≤11 core genome SNP [Single Nucleotide Polymorphism] differences) between case and epidemiologically-linked carrier isolates; all were genotypes prevalent in 1980s Santiago. A cluster of four additional autochthonous cases un-linked to a carrier was identified based on genomic identity (0-1 SNPs). Travel/immigration isolate genotypes were typical for the countries of travel/immigration. CONCLUSIONS: Although autochthonous typhoid cases in Santiago are currently rare, 5/16 such cases (31.3%) were linked to elderly chronic carriers identified among household contacts of cases.

Evaluation of Three Candidate Live-Attenuated Salmonella enterica Serovar Typhimurium Vaccines to Prevent Non-Typhoidal Salmonella Infection in an Infant Mouse Model.

Nontyphoidal Salmonella enterica (NTS) is a leading cause of foodborne illness worldwide, including in the United States, where infants show the highest incidence amongst all age groups. S. enterica serovar Typhimurium is one of the most frequently isolated serovars from NTS infections. We have developed several candidate live-attenuated S. Typhimurium vaccines to prevent NTS infection. The goal of the current study was to assess three live S. Typhimurium vaccine strains (CVD 1921, CVD 1921 ∆htrA and CVD 1926, which have two, three and four gene deletions, respectively) with various levels of reactogenicity and immunogenicity in infant BALB/c mice to predict how they would perform following peroral immunization of infants. We first tested intranasal immunization of 14-day-old mice with three doses delivered at 1-week intervals and evaluated antibody responses and protection against lethal infection with wild-type S. Typhimurium. The vaccines were administered to 14-day-old mice via the peroral route at 1- or 2-week intervals and to 28-day-old mice at 2-week intervals. The three vaccine strains were immunogenic following intranasal immunization of infant mice with vaccine efficacies of 80% (CVD 1921), 63% (CVD 1921 ∆htrA) and 31% (CVD 1926). In contrast, peroral immunization of 14-day-old mice yielded much poorer protection against lethal infection and only immunization of 28-day-old mice at 2-week intervals showed similar protective capacity as intranasal administration (CVD 1921: 83%, CVD 1921 ∆htrA: 43% and CVD 1926: 58%). CVD 1921 was consistently more protective than both CVD 1921 ∆htrA and CVD 1926, regardless of the route of vaccination, immunization schedule and age of mice. Anti-LPS serum IgG responses were similar between the three strains and did not correlate with protection. Due to previously observed reactogenicity of CVD 1921, CVD 1921 ∆htrA and CVD 1926 are our preferred vaccines, but these data show that further improvements would need to be made to achieve suitable protection in young infants when using peroral immunization.

Durable Anti-Vi IgG and IgA Antibody Responses in 15-Month-Old Children Vaccinated With Typhoid Conjugate Vaccine in Burkina Faso.

We assessed anti-Vi IgG/IgA responses to typhoid conjugate vaccine (TCV) in children enrolled in a double-blind randomized controlled, phase 2 trial in Burkina Faso. Anti-Vi IgG seroconversion and anti-Vi IgA titers were higher in TCV than control recipients at 30-35 months post-vaccination. TCV induces durable immunity in Burkinabe children vaccinated at 15 months.

COVID-19 booster vaccination during pregnancy enhances maternal binding and neutralizing antibody responses and transplacental antibody transfer to the newborn.

The immune response to COVID-19 booster vaccinations during pregnancy for mothers and their newborns and the functional response of vaccine-induced antibodies against Omicron variants are not well characterized. We conducted a prospective, multicenter cohort study of participants vaccinated during pregnancy with primary or booster mRNA COVID-19 vaccines from July 2021 to January 2022 at 9 academic sites. We determined SARS-CoV-2 binding and live virus and pseudovirus neutralizing antibody (nAb) titers pre- and post-vaccination, and at delivery for both maternal and infant participants. Immune responses to ancestral and Omicron BA.1 SARS-CoV-2 strains were compared between primary and booster vaccine recipients in maternal sera at delivery and in cord blood, after adjusting for days since last vaccination. A total of 240 participants received either Pfizer or Moderna mRNA vaccine during pregnancy (primary 2-dose series: 167; booster dose: 73). Booster vaccination resulted in significantly higher binding and nAb titers, including to the Omicron BA.1 variant, in maternal serum at delivery and in cord blood compared to a primary 2-dose series (range 0.44-0.88 log10 higher, p < 0.0001 for all comparisons). Live virus nAb to Omicron BA.1 were present at delivery in 9 % (GMT ID50 12.7) of Pfizer and 22 % (GMT ID50 14.7) of Moderna primary series recipients, and in 73 % (GMT ID50 60.2) of mRNA boosted participants (p < 0.0001), although titers were significantly lower than to the D614G strain. Transplacental antibody transfer was efficient for all regimens with median transfer ratio range: 1.55-1.77 for IgG, 1.00-1.78 for live virus nAb and 1.79-2.36 for pseudovirus nAb. COVID-19 mRNA vaccination during pregnancy elicited robust immune responses in mothers and efficient transplacental antibody transfer to the newborn. A booster dose during pregnancy significantly increased maternal and cord blood binding and neutralizing antibody levels, including against Omicron BA.1. Findings support the use of a booster dose of COVID-19 vaccine during pregnancy.

Co-culturing Human Intestinal Enteroid Monolayers with Innate Immune Cells.

The coordinated interaction between the intestinal epithelium and immune cells is required to maintain proper barrier function and mucosal host defenses to the harsh external environment of the gut lumen. Complementary to in vivo models, there is a need for practical and reproducible in vitro models that employ primary human cells to confirm and advance our understanding of mucosal immune responses under physiologic and pathophysiologic conditions. Here we describe the methods to co-culture human intestinal stem cell-derived enteroids grown as confluent monolayers on permeable supports with primary human innate immune cells (e.g., monocyte-derived macrophages and polymorphonuclear neutrophils). This co-culture model reconstructs the cellular framework of the human intestinal epithelial-immune niche with distinct apical and basolateral compartments to recreate host responses to luminal and submucosal challenges, respectively. Enteroid-immune co-cultures enable multiple outcome measures to interrogate important biological processes such as epithelial barrier integrity, stem cell biology, cellular plasticity, epithelial-immune cells crosstalk, immune cell effector functions, changes in gene expression (i.e., transcriptomic, proteomic, epigenetic), and host-microbiome interactions.

A Novel Shigella O-Polysaccharide-IpaB Conjugate Vaccine Elicits Robust Antibody Responses and Confers Protection against Multiple Shigella Serotypes.

Shigella is responsible for high burdens of diarrhea and dysentery globally. Children living in areas of endemicity are the most affected, and currently, there are no licensed vaccines to prevent shigellosis. Vaccine approaches have traditionally targeted the bacterial lipopolysaccharide as a protective antigen. Shigella O-polysaccharide (OPS) conjugated to recombinant Pseudomonas aeruginosa exotoxin A (rEPA) or tetanus toxoid (TT) is advanced in clinical evaluation. Adequate efficacy of these vaccines, particularly in the infant target group, remains to be demonstrated. A major limitation of the OPS-glycoconjugate concept is its limited coverage, since immunity to the O antigen is serotype specific, and there are multiple disease-causing serotypes. Another concern is the use of protein carriers already included in multiple other childhood vaccines. This study reports a novel Shigella OPS conjugate vaccine that uses the Shigella invasion plasmid antigen B (IpaB) as the carrier protein. IpaB is a virulence factor component of the Shigella type III secretion system and highly conserved among Shigella serotypes. It is robustly immunogenic and a protective antigen. IpaB and IpaB containing nonnative amino acids (nnAA) were produced at large scale using cell-free protein synthesis. Incorporation of nnAA enabled site-specific conjugation of IpaB to Shigella flexneri 2a OPS using click chemistry, yielding OPS-IpaB glycoconjugate. Parenteral immunization of mice with the OPS-IpaB vaccine resulted in high levels of OPS- and IpaB-specific serum IgG and robust protection against lethal S. flexneri 2a or Shigella sonnei challenge. The OPS-IpaB vaccine is a promising new vaccine candidate with the capacity to confer broad protection against clinically relevant Shigella serotypes. IMPORTANCE Diarrhea caused by Shigella species results in long-term disability and mortality globally, disproportionally affecting younger children living in poor countries. Although it is treatable by antibiotics, the rapid and widespread emergence of resistant strains and the highly contagious nature of the disease compel the development of preventive tools. Currently, several Shigella OPS conjugate vaccines are being evaluated in clinical studies, but these rely exclusively on immunity against the bacterial O antigen, which limits their coverage to only the immunizing serotype; multivalent vaccines are needed to protect against the most prevalent serotypes. This is the first report of a novel Shigella OPS-conjugate vaccine that uses Shigella IpaB as a carrier and protective antigen. This vaccine, administered parenterally, elicited robust immunity and protected mice against lethal infection by S. flexneri 2a or S. sonnei. The OPS-IpaB vaccine is a promising candidate for evaluation in vulnerable populations.

Histo-Blood Group Antigen Null Phenotypes Associated With a Decreased Risk of Clinical Rotavirus Vaccine Failure Among Children <2 Years of Age Participating in the Vaccine Impact on Diarrhea in Africa (VIDA) Study in Kenya, Mali, and the Gambia.

BACKGROUND: Previously studied risk factors for rotavirus vaccine failure have not fully explained reduced rotavirus vaccine effectiveness in low-income settings. We assessed the relationship between histo-blood group antigen (HBGA) phenotypes and clinical rotavirus vaccine failure among children <2 years of age participating in the Vaccine Impact on Diarrhea in Africa Study in 3 sub-Saharan African countries. METHODS: Saliva was collected and tested for HBGA phenotype in children who received rotavirus vaccine. The association between secretor and Lewis phenotypes and rotavirus vaccine failure was examined overall and by infecting rotavirus genotype using conditional logistic regression in 218 rotavirus-positive cases with moderate-to-severe diarrhea and 297 matched healthy controls. RESULTS: Both nonsecretor and Lewis-negative phenotypes (null phenotypes) were associated with decreased rotavirus vaccine failure across all sites (matched odds ratio, 0.30 [95% confidence interval: 0.16-0.56] or 0.39 [0.25-0.62], respectively]. A similar decrease in risk against rotavirus vaccine failure among null HBGA phenotypes was observed for cases with P[8] and P[4] infection and their matched controls. While we found no statistically significant association between null HBGA phenotypes and vaccine failure among P[6] infections, the matched odds ratio point estimate for Lewis-negative individuals was >4. CONCLUSIONS: Our study demonstrated a significant relationship between null HBGA phenotypes and decreased rotavirus vaccine failure in a population with P[8] as the most common infecting genotype. Further studies are needed in populations with a large burden of P[6] rotavirus diarrhea to understand the role of host genetics in reduced rotavirus vaccine effectiveness.

H3N2 influenza hemagglutination inhibition method qualification with data driven statistical methods for human clinical trials.

Introduction: Hemagglutination inhibition (HAI) antibody titers to seasonal influenza strains are important surrogates for vaccine-elicited protection. However, HAI assays can be variable across labs, with low sensitivity across diverse viruses due to lack of standardization. Performing qualification of these assays on a strain specific level enables the precise and accurate quantification of HAI titers. Influenza A (H3N2) continues to be a predominant circulating subtype in most countries in Europe and North America since 1968 and is thus a focus of influenza vaccine research. Methods: As a part of the National Institutes of Health (NIH)-funded Collaborative Influenza Vaccine Innovation Centers (CIVICs) program, we report on the identification of a robust assay design, rigorous statistical analysis, and complete qualification of an HAI assay using A/Texas/71/2017 as a representative H3N2 strain and guinea pig red blood cells and neuraminidase (NA) inhibitor oseltamivir to prevent NA-mediated agglutination. Results: This qualified HAI assay is precise (calculated by the geometric coefficient of variation (GCV)) for intermediate precision and intra-operator variability, accurate calculated by relative error, perfectly linear (slope of -1, R-Square 1), robust (<25% GCV) and depicts high specificity and sensitivity. This HAI method was successfully qualified for another H3N2 influenza strain A/Singapore/INFIMH-16-0019/2016, meeting all pre-specified acceptance criteria. Discussion: These results demonstrate that HAI qualification and data generation for new influenza strains can be achieved efficiently with minimal extra testing and development. We report on a qualified and adaptable influenza serology method and analysis strategy to measure quantifiable HAI titers to define correlates of vaccine mediated protection in human clinical trials.

Distinct in vitro and in vivo neutralization profiles of monoclonal antibodies elicited by the receptor binding domain of the ancestral SARS-CoV-2.

Broadly neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are sought to curb coronavirus disease 2019 (COVID-19) infections. Here we produced and characterized a set of mouse monoclonal antibodies (mAbs) specific for the ancestral SARS-CoV-2 receptor binding domain (RBD). Two of them, 17A7 and 17B10, were highly potent in microneutralization assay with 50% inhibitory concentration (IC50 ) ≤135 ng/mL against infectious SARS-CoV-2 variants, including G614, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Kappa, Lambda, B.1.1.298, B.1.222, B.1.5, and R.1. Both mAbs (especially 17A7) also exhibited strong in vivo efficacy in protecting K18-hACE2 transgenic mice from the lethal infection with G614, Alpha, Beta, Gamma, and Delta viruses. Structural analysis indicated that 17A7 and 17B10 target the tip of the receptor binding motif in the RBD-up conformation. A third RBD-reactive mAb (3A6) although escaped by Beta and Gamma, was highly effective in cross-neutralizing Delta and Omicron BA.1 variants in vitro and in vivo. In competition experiments, antibodies targeting epitopes similar to these 3 mAbs were rarely enriched in human COVID-19 convalescent sera or postvaccination sera. These results are helpful to inform new antibody/vaccine design and these mAbs can be useful tools for characterizing SARS-CoV-2 variants and elicited antibody responses.

Enhanced virulence and waning vaccine-elicited antibodies account for breakthrough infections caused by SARS-CoV-2 delta and beyond.

Here we interrogate the factors responsible for SARS-CoV-2 breakthrough infections in a K18-hACE2 transgenic mouse model. We show that Delta and the closely related Kappa variant cause viral pneumonia and severe lung lesions in K18-hACE2 mice. Human COVID-19 mRNA post-vaccination sera after the 2nd dose are significantly less efficient in neutralizing Delta/Kappa than early 614G virus in vitro and in vivo. By 5 months post-vaccination, ≥50% of donors lack detectable neutralizing antibodies against Delta and Kappa and all mice receiving 5-month post-vaccination sera die after the lethal challenges. Although a 3rd vaccine dose can boost antibody neutralization against Delta in vitro and in vivo, the mean log neutralization titers against the latest Omicron subvariants are 1/3-1/2 of those against the original 614D virus. Our results suggest that enhanced virulence, greater immune evasion, and waning of vaccine-elicited protection account for SARS-CoV-2 variants caused breakthrough infections.

Dynamics of the Gut Microbiome in Shigella-Infected Children during the First Two Years of Life.

Shigella continues to be a major contributor to diarrheal illness and dysentery in children younger than 5 years of age in low- and middle-income countries. Strategies for the prevention of shigellosis have focused on enhancing adaptive immunity. The interaction between Shigella and intrinsic host factors, such as the microbiome, remains unknown. We hypothesized that Shigella infection would impact the developing microbial community in infancy and, conversely, that changes in the gastrointestinal microbiome may predispose infections. To test this hypothesis, we characterized the gastrointestinal microbiota in a longitudinal birth cohort from Malawi that was monitored for Shigella infection using 16S rRNA amplicon sequencing. Children with at least one Shigella quantitative polymerase chain reaction (qPCR) positive sample during the first 2 years of life (cases) were compared to uninfected controls that were matched for sex and age. Overall, the microbial species diversity, as measured by the Shannon diversity index, increased over time, regardless of case status. At early time points, the microbial community was dominated by Bifidobacterium longum and Escherichia/Shigella. A greater abundance of Prevotella 9 and Bifidobacterium kashiwanohense was observed at 2 years of age. While no single species was associated with susceptibility to Shigella infection, significant increases in Lachnospiraceae NK4A136 and Fusicatenibacter saccharivorans were observed following Shigella infection. Both taxa are in the family Lachnospiraceae, which are known short-chain fatty acid producers that may improve gut health. Our findings identified temporal changes in the gastrointestinal microbiota associated with Shigella infection in Malawian children and highlight the need to further elucidate the microbial communities associated with disease susceptibility and resolution. IMPORTANCE Shigella causes more than 180 million cases of diarrhea globally, mostly in children living in poor regions. Infection can lead to severe health impairments that reduce quality of life. There is increasing evidence that disruptions in the gut microbiome early in life can influence susceptibility to illnesses. A delayed or impaired reconstitution of the microbiota following infection can further impact overall health. Aiming to improve our understanding of the interaction between Shigella and the developing infant microbiome, we investigated changes in the gut microbiome of Shigella-infected and uninfected children over the course of their first 2 years of life. We identified species that may be involved in recovery from Shigella infection and in driving the microbiota back to homeostasis. These findings support future studies into the elucidation of the interaction between the microbiota and enteric pathogens in young children and into the identification of potential targets for prevention or treatment.

Systems approach to define humoral correlates of immunity to Shigella.

Shigella infection is the second leading cause of death due to diarrheal disease in young children worldwide. With the rise of antibiotic resistance, initiatives to design and deploy a safe and effective Shigella vaccine are urgently needed. However, efforts to date have been hindered by the limited understanding of immunological correlates of protection against shigellosis. We applied systems serology to perform a comprehensive analysis of Shigella-specific antibody responses in sera obtained from volunteers before and after experimental infection with S. flexneri 2a in a series of controlled human challenge studies. Polysaccharide-specific antibody responses are infrequent prior to infection and evolve concomitantly with disease severity. In contrast, pre-existing antibody responses to type 3 secretion system proteins, particularly IpaB, consistently associate with clinical protection from disease. Linked to particular Fc-receptor binding patterns, IpaB-specific antibodies leverage neutrophils and monocytes, and complement and strongly associate with protective immunity. IpaB antibody-mediated functions improve with a subsequent rechallenge resulting in complete clinical protection. Collectively, our systems serological analyses indicate protein-specific functional correlates of immunity against Shigella in humans.

Mucosal Immune Profiles Associated with Diarrheal Disease Severity in Shigella- and Enteropathogenic Escherichia coli-Infected Children Enrolled in the Global Enteric Multicenter Study.

Enteropathogenic Escherichia coli (EPEC) and Shigella are etiologic agents of diarrhea in children <5 years old living in resource-poor countries. Repeated bouts of infection lead to lifelong morbidity and even death. The goal of this study was to characterize local mucosal immune responses in Shigella- and EPEC-infected children <5 years of age with moderate to severe diarrhea (MSD) enrolled in the Global Enteric Multicenter Study (GEMS). We hypothesized that infection with each of these pathogens would induce distinct gut mucosal immune profiles indicative of disease etiology and severity. To test this hypothesis, innate and adaptive immune markers were measured in stools from children with diarrhea due to EPEC, Shigella, or other organisms and in children who had no diarrhea. Shigella-positive diarrhea evoked robust proinflammatory and TH1/TH2 cytokine responses compared to diarrhea caused by EPEC or other organisms, with the exception of interleukin 5 (IL-5), which was associated with EPEC infection. The presence of IL-1ß, IL-4, IL-16, and tumor necrosis factor beta (TNF-ß) was associated with the absence of dysentery. EPEC-positive diarrhea evoked high levels of IL-1ß, vascular endothelial growth factor (VEGF), and IL-10. Granulocyte-macrophage colony-stimulating factor (GM-CSF) had opposing roles in disease severity, being associated with absence of diarrhea in EPEC-infected children and with dysenteric Shigella infection. High levels of antigen-specific antibodies were detected in the controls and children with Shigella without dysentery, which suggests a protective role against severe disease. In summary, this study identified distinct local immune responses associated with two clinically relevant diarrheagenic pathogens, Shigella and EPEC, in children and identified protective immune phenotypes that can inform the development of preventive measures. IMPORTANCE Shigella and enteropathogenic Escherichia coli are primary agents of moderate to severe diarrhea in children <5 years of age living in resource-poor countries. Repeated bouts of illness lead to lifelong health impairment and even death. Aiming to understand the local host immunity to these pathogens in relation to disease prognosis and to identify prophylaxis and therapeutic targets, we investigated innate and adaptive immune profiles in stools from children infected with EPEC with and without diarrhea, Shigella with and without dysentery, and controls in well characterized clinical samples obtained during the Global Enteric Multicenter Study. For the first time, we report pathogen-specific mucosal immune profiles associated with severity or absence of disease in children <5 years of age that can inform prevention and treatment efforts.

Safety and immunogenicity of a typhoid conjugate vaccine among children aged 9 months to 12 years in Malawi: a nested substudy of a double-blind, randomised controlled trial.

BACKGROUND: Typhoid fever is a substantial public health problem in Africa, yet there are few clinical trials of typhoid conjugate vaccine (TCV). We assessed immunogenicity and safety of Typbar TCV in Malawi. METHODS: This substudy was nested within a phase 3, double-blind, parallel design, randomised controlled trial of TCV in children from Ndirande Health Centre in Ndirande township, Blantyre, Malawi. To be eligible, participants had to be aged between 9 months and 12 years with no known immunosuppression or chronic health conditions, including HIV or severe malnutrition; eligible participants were enrolled into three strata of approximately 200 children (9-11 months, 1-5 years, and 6-12 years), randomly assigned (1:1) to receive TCV or control (meningococcal serogroup A conjugate vaccine [MCV-A]) intramuscularly. Serum was collected before vaccination and at 28 days and 730-1035 days after vaccination to measure anti-Vi antibodies by ELISA. Because of COVID-19, day 730 visits were extended up to 1035 days. This nested substudy evaluated reactogenicity, safety, and immunogenicity by age stratum. Safety outcomes, analysed in the intention-to-treat population, included solicited adverse events within 7 days of vaccination (assessed on 3 separate days) and unsolicited adverse events within 28 days of vaccination. This trial is registered with ClinicalTrials.gov, NCT03299426. FINDINGS: Between Feb 22 and Sept 6, 2018, 664 participants were screened, and 631 participants were enrolled and randomly assigned (320 to the TCV group and 311 to the MCV-A group). 305 participants in the TCV group and 297 participants in the MCV-A group were vaccinated. Among TCV recipients, anti-Vi IgG geometric mean titres increased more than 500 times from 4·2 ELISA units (EU)/mL (95% CI 4·0-4·4) at baseline to 2383·7 EU/mL (2087·2-2722·3) at day 28, then decreased to 48·0 EU/mL (39·9-57·8) at day 730-1035, remaining more than 11 times higher than baseline. Among MCV-A recipients, anti-Vi IgG titres remained unchanged: 4·3 EU/mL (4·0-4·5) at baseline, 4·4 EU/mL (4·0-4·7) on day 28, and 4·6 EU/mL (4·2-5·0) on day 730-1035. TCV and MCV-A recipients had similar solicited local (eight [3%] of 304, 95% CI 1·3-5·1 and three [1%] of 293, 0·4-3·0) and systemic (27 [9%] of 304, 6·2-12·6 and 27 [9%] of 293, 6·4-13·1) reactogenicity. Related unsolicited adverse events occurred similarly in TCV and MCV-A recipients in eight (3%) of 304 (1·3-5·1) and eight (3%) of 293 (1·4-5·3). INTERPRETATION: This study provides evidence of TCV safety, tolerability, and immunogenicity up to 730-1035 days in Malawian children aged 9 months to 12 years. FUNDING: Bill & Melinda Gates Foundation.

Epithelial and Neutrophil Interactions and Coordinated Response to Shigella in a Human Intestinal Enteroid-Neutrophil Coculture Model.

Polymorphonuclear neutrophils (PMN) are recruited to the gastrointestinal mucosa in response to inflammation, injury, and infection. Here, we report the development and the characterization of an ex vivo tissue coculture model consisting of human primary intestinal enteroid monolayers and PMN, and a mechanistic interrogation of PMN-epithelial cell interaction and response to Shigella, a primary cause of childhood dysentery. Cellular adaptation and tissue integration, barrier function, PMN phenotypic and functional attributes, and innate immune responses were examined. PMN within the enteroid monolayers acquired a distinct activated/migratory phenotype that was influenced by direct epithelial cell contact as well as by molecular signals. Seeded on the basal side of the intestinal monolayer, PMN were intercalated within the epithelial cells and moved paracellularly toward the apical side. Cocultured PMN also increased basal secretion of interleukin 8 (IL-8). Shigella added to the apical surface of the monolayers evoked additional PMN phenotypic adaptations, including increased expression of cell surface markers associated with chemotaxis and cell degranulation (CD47, CD66b, and CD88). Apical Shigella infection triggered rapid transmigration of PMN to the luminal side, neutrophil extracellular trap (NET) formation, and bacterial phagocytosis and killing. Shigella infection modulated cytokine production in the coculture; apical monocyte chemoattractant protein (MCP-1), tumor necrosis factor alpha (TNF-α), and basolateral IL-8 production were downregulated, while basolateral IL-6 secretion was increased. We demonstrated, for the first time, PMN phenotypic adaptation and mobilization and coordinated epithelial cell-PMN innate response upon Shigella infection in the human intestinal environment. The enteroid monolayer-PMN coculture represents a technical innovation for mechanistic interrogation of gastrointestinal physiology, host-microbe interaction, innate immunity, and evaluation of preventive/therapeutic tools. IMPORTANCE Studies of mucosal immunity and microbial host cell interaction have traditionally relied on animal models and in vitro tissue culture using immortalized cancer cell lines, which yield nonphysiological and often unreliable results. Herein, we report the development and characterization of an ex vivo enteroid-PMN coculture consisting of normal human intestinal epithelium and a mechanistic interrogation of PMN and epithelial cell interaction and function in the context of Shigella infection. We demonstrated tissue-driven phenotypic and functional adaptation of PMN and a coordinated epithelial cell and PMN response to Shigella, a primary cause of dysentery in young children in the developing world.