IgA Responses Research Articles

Oral subunit SARS-CoV-2 vaccine induces systemic neutralizing IgG, IgA and cellular immune responses and can boost neutralizing antibody responses primed by an injected vaccine

The rapid spread of the COVID-19 pandemic, with its devastating medical and economic impacts, triggered an unprecedented race toward development of effective vaccines. The commercialized vaccines are parenterally administered, which poses logistic challenges, while adequate protection at the mucosal sites of virus entry is questionable. Furthermore, essentially all vaccine candidates target the viral spike (S) protein, a surface protein that undergoes significant antigenic drift. This work aimed to develop an oral multi-antigen SARS-CoV-2 vaccine comprised of the receptor binding domain (RBD) of the viral S protein, two domains of the viral nucleocapsid protein (N), and heat-labile enterotoxin B (LTB), a potent mucosal adjuvant. The humoral, mucosal and cell-mediated immune responses of both a three-dose vaccination schedule and a heterologous subcutaneous prime and oral booster regimen were assessed in mice and rats, respectively. Mice receiving the oral vaccine compared to control mice showed significantly enhanced post-dose-3 virus-neutralizing antibody, anti-S IgG and IgA production and N-protein-stimulated IFN-γ and IL-2 secretion by T cells. When administered as a booster to rats following parenteral priming with the viral S1 protein, the oral vaccine elicited markedly higher neutralizing antibody titres than did oral placebo booster. A single oral booster following two subcutaneous priming doses elicited serum IgG and mucosal IgA levels similar to those raised by three subcutaneous doses. In conclusion, the oral LTB-adjuvanted multi-epitope SARS-CoV-2 vaccine triggered versatile humoral, cellular and mucosal immune responses, which are likely to provide protection, while also minimizing technical hurdles presently limiting global vaccination, whether by priming or booster programs.

Protective Effects of Zinc on Salmonella Invasion, Intestinal Morphology and Immune Response of Young Pigeons Infected with Salmonella enterica Serovar Typhimurium.

The study aimed to determine the effects of orally supplemental zinc on body weight, Salmonella invasion, serum IgA, intestinal histomorphology, and immune response of Salmonella enterica serovar Typhimurium (S. typhimurium)-challenged young pigeons. A total of 72 healthy White King pigeons (25days old) with similar weight were randomly assigned to 3 treatments with six replicate cages. The 3 treatments were unchallenged, S. typhimurium-challenged, and S. typhimurium-challenged orally supplemented with 1mg zinc per bird. Salmonella infection decreased (P < 0.05) the body weight, the bursa index, the serum IgA content, and the villus height/crypt depth ratio in the ileum, but increased the neutrophil proportion (P < 0.001) and the mRNA expressions of IL-1β and IL-8 in the jejunum (P < 0.05). Orally supplemental zinc reduced (P = 0.007) the bacterial load in the liver and improved (P < 0.05) the body weight, the bursa index, the serum IgA content, the villus height/crypt depth ratio, and the NOD-like receptor family pyrin domain containing 3 (NLRP3) protein expression, as well as tended to increase (P = 0.064) the protein abundance of caspase-1 of the jejunum, but did not alleviate the high level of neutrophil proportion and IL-1β mRNA expression of the jejunum (P > 0.05). The results indicated that oral zinc supplementation improved the intestinal mucosal morphology and enhanced the immune response, as well as activated caspase-1-dependent cell pyroptosis pathways in the jejunal epithelium, thereby restricting Salmonella invasion of the challenged young pigeons.

B cell class switching in intestinal immunity in health and disease.

The gastrointestinal tract is colonized by trillions of commensal microorganisms that collectively form the microbiome and make essential contributions to organism homeostasis. The intestinal immune system must tolerate these beneficial commensals, whilst preventing pathogenic organisms from systemic spread. Humoral immunity plays a key role in this process, with large quantities of immunoglobulin (Ig)A secreted into the lumen on a daily basis, regulating the microbiome and preventing bacteria from encroaching on the epithelium. However, there is an increasing appreciation of the role of IgG antibodies in intestinal immunity, including beneficial effects in neonatal immune development, pathogen and tumour resistance, but also of pathological effects in driving chronic inflammation in inflammatory bowel disease (IBD). These antibody isotypes differ in effector function, with IgG exhibiting more proinflammatory capabilities compared with IgA. Therefore, the process that leads to the generation of different antibody isotypes, class‐switch recombination (CSR), requires careful regulation and is orchestrated by the immunological cues generated by the prevalent local challenge. In general, an initiating signal such as CD40 ligation on B cells leads to the induction of activation‐induced cytidine deaminase (AID), but a second cytokine‐mediated signal determines which Ig heavy chain is expressed. Whilst the cytokines driving intestinal IgA responses are well‐studied, there is less clarity on how IgG responses are generated in the intestine, and how these cues might become dysfunctional in IBD. Here, we review the key mechanisms regulating class switching to IgA vs IgG in the intestine, processes that could be therapeutically manipulated in infection and IBD.

Construction of the Enterococcal Strain Expressing Immunogenic Fragment of SARS-Cov-2 Virus

Contemporary SARS-Cov-2 pandemic, besides its dramatic global influence on the human race including health care systems, economies, and political decisions, opened a window for the global experiment with human vaccination employing novel injectable vaccines providing predominantly specific IgG response with little knowledge of their impact on the mucosal immunity. However, it is widely accepted that protection against the pathogens at the gates of the infection - on mucosal surfaces—predominantly rely on an IgA response. Some genetically modified bacteria, including probiotics, represent attractive vehicles for oral or nasal mucosal delivery of therapeutic molecules. Probiotic-based vaccines for mucous membranes are easy to produce in large quantities; they have low cost, provide quite a long T-cell memory, and gut IgA response to oral vaccines is highly synchronized and strongly oligoclonal. Here we present a study demonstrating construction of the novel SARS-Cov-2 vaccine candidate employing the gene fragment of S1 SARS-Cov-2 gene. This DNA fragment was inserted in frame into major pili protein gene with d2 domain of enterococcal operon encoding for pili. The DNA sequencing proved the presence of the insert in enterococcal genome. RNA transcription, immunoprecipitation, and immune electron microscopy with human sera obtained from the SARS-Cov-2 patients demonstrated expression of SARS-Cov-2 antigens in bacteria. Taken together the data obtained allowed considering this genetically modified probiotic strain as an interesting candidate for vaccine against SARS-Cov-2.

Construction of the Enterococcal Strain Expressing Immunogenic Fragment of SARS-Cov-2 Virus

Contemporary SARS-Cov-2 pandemic, besides its dramatic global influence on the human race including health care systems, economies, and political decisions, opened a window for the global experiment with human vaccination employing novel injectable vaccines providing predominantly specific IgG response with little knowledge of their impact on the mucosal immunity. However, it is widely accepted that protection against the pathogens at the gates of the infection - on mucosal surfaces—predominantly rely on an IgA response. Some genetically modified bacteria, including probiotics, represent attractive vehicles for oral or nasal mucosal delivery of therapeutic molecules. Probiotic-based vaccines for mucous membranes are easy to produce in large quantities; they have low cost, provide quite a long T-cell memory, and gut IgA response to oral vaccines is highly synchronized and strongly oligoclonal. Here we present a study demonstrating construction of the novel SARS-Cov-2 vaccine candidate employing the gene fragment of S1 SARS-Cov-2 gene. This DNA fragment was inserted in frame into major pili protein gene with d2 domain of enterococcal operon encoding for pili. The DNA sequencing proved the presence of the insert in enterococcal genome. RNA transcription, immunoprecipitation, and immune electron microscopy with human sera obtained from the SARS-Cov-2 patients demonstrated expression of SARS-Cov-2 antigens in bacteria. Taken together the data obtained allowed considering this genetically modified probiotic strain as an interesting candidate for vaccine against SARS-Cov-2.

Abrocitinib 100 mg versus 200 mg for atopic dermatitis: a meta-analysis of randomized controlled trials.

It is elusive to compare the efficacy and safety of abrocitinib 100 mg versus 200 mg once daily in patients with atopic dermatitis. This meta-analysis aims to explore the influence of abrocitinib 100 mg versus 200 mg on the treatment of atopic dermatitis. Several databases including PubMed, EMbase, Web of science, EBSCO, and Cochrane library were systematically searched through July 2021. We included randomized controlled trials (RCTs) assessing the effect of abrocitinib 100 mg versus 200 mg for patients with atopic dermatitis. Four RCTs were included in the meta-analysis. Compared with abrocitinib 100 mg for atopic dermatitis, abrocitinib 200 mg had a remarkably positive impact on IGA response (OR = 1.78; 95% CI: 1.39-2.28; p < 0.00001), EASI-75 (OR = 2.03; 95% CI: 1.60-2.57; p < 0.00001), NRS response (OR = 1.97; 95% CI: 1.27-3.08; p = 0.003), and adverse events (OR = 1.43; 95% CI: 1.11-1.84; p = 0.005), but it showed no obvious influence on serious adverse events (OR = 0.59; 95% CI: 0.25-1.37; p = 0.22). Abrocitinib 200 mg is better than abrocitinib 100 mg for the treatment of atopic dermatitis.

Dynamics of Gut Microbiome, IgA Response and Plasma Metabolome in Development of Pediatric Celiac Disease

Dynamics of Gut Microbiome, IgA Response and Plasma Metabolome in Development of Pediatric Celiac Disease

Application of Antimicrobial Peptide LL-37 as an Adjuvant for Middle East Respiratory Syndrome-Coronavirus Antigen Induces an Efficient Protective Immune Response Against Viral Infection After Intranasal Immunization.

The human antimicrobial peptide LL-37 has chemotactic and modulatory activities in various immune cells, including dendritic cells. Because of its characteristics, LL-37 can be considered an adjuvant for vaccine development. In this study, we confirmed the possible adjuvant activity of LL-37 in mucosal vaccine development against Middle East respiratory syndrome-coronavirus (MERS-CoV) by means of intranasal immunization in C57BL/6 and human dipeptidyl peptidase 4 (hDPP4)-transgenic (hDPP4-Tg) mice. Intranasal immunization using the receptor-binding domain (RBD) of MERS-CoV spike protein (S-RBD) recombined with LL-37 (S-RBD-LL-37) induced an efficient mucosal IgA and systemic IgG response with virus-neutralizing activity, compared with S-RBD. Ag-specific CTL stimulation was also efficiently induced in the lungs of mice that had been intranasally immunized with S-RBD-LL-37, compared with S-RBD. Importantly, intranasal immunization of hDPP4-Tg mice with S-RBD-LL-37 led to reduced immune cell infiltration into the lungs after infection with MERS-CoV. Finally, intranasal immunization of hDPP4-Tg mice with S-RBD-LL-37 led to enhanced protective efficacy, with increased survival and reduced body weight loss after challenge infection with MERS-CoV. Collectively, these results suggest that S-RBD-LL-37 is an effective intranasal vaccine candidate molecule against MERS-CoV infection.

Generation of Bacillus subtilis displaying alpha-toxin HlaH35LH48L fused with CotB and CotG, and studying the immune response in mice

Introduction: The Bacillus subtilis spore is considered to be a powerful vehicle for surface display and antigen delivery. Being safe and widely used for the purpose of oral bacteriotherapy in humans and animals, B. subtilis spore has potential in the domain of needle-free vaccine development. The spores themselves also behave as an adjuvant. This study aims to generate B. subtilis spores expressing mutant staphylococcal alpha-toxin HlaH35LH48L on the surface and to study their ability to evoke a specific immune response in mice. Methods: Vectors carrying the hlaH35LH48L gene fused with the coding genes of anchor proteins, cotB and cotG, were cloned in E. coli before being transformed into B. subtilis. The generation of the B. subtilis new strains via chromosomal integration was confirmed by PCR. Sporulation was observed under the microscope. The expression of the target protein on the spore surface was determined by sporeELISA. The level of IgG in the serum and IgA in the feces of Swiss mice were analyzed using ELISA to learn about the immune response against the B. subtilis spore administrated via the oral route. Results: The PCR products of an expected size on agarose gel electrophoresis showed successful integration, resulting in the construction of new B. subtilis strains BsHT2331 and BsHT2334. sporeELISA analysis detected a significant HlaH35LH48L expression on the spore surface. The BsHT2331 spores (CotB-HlaH35LH48L) triggered a constant increase in the IgG and IgA levels in mice after three doses as 5.5-fold and 2.5-fold higher than the pre-immunization, respectively (p-value &lt; 0.0001). Meanwhile, the group of mice orally administrated with BsHT2334 (CotG-HlaH35LH48L) showed a notable IgG titer but minor IgA response. Conclusion: The results concluded that the construction of two new strains BsHT2331 and BsHT2334 that used spore coat proteins CotB and CotG to display mutant alpha-toxin HlaH35LH48L on the B. subtilis spore surface was successful. It provided an assessment of the ability of B. subtilis spores to stimulate specific antibody production in mice.

Integrin CD11b provides a new marker of pre-germinal center IgA+ B cells in murine Peyer's patches.

Activated B cells can enter germinal centers (GCs) for affinity maturation to produce high-affinity antibodies. However, which activated B cells will enter GCs remains unknown. Here, we found a small population of CD11b+IgA+ B cells located outside of GCs in murine Peyer’s patches (PPs). After injection of the CD11b+IgA+ PP B cells into a PP of a recipient mouse, they entered GCs forty hours later. They expressed GC surface markers and pre-GC B cell genes, suggesting that CD11b provides a novel surface marker of pre-GC IgA+ B cells in murine PPs. Furthermore, independently of dendritic cell activation, CD11b expression on B cells can be induced by bacterial antigens, such as pam3CSK4 and heat-killed Escherichia coli in vitro. In addition, mice orally administered with pam3CSK4 or heat-killed E. coli increased the number of PP GC B cells within two days, and enhanced the mucosal antigen-specific IgA response. Our results demonstrate that the induction of CD11b on B cells is a promising marker for selecting an effective mucosal vaccine adjuvant.

Surplus sterols suppress Salmonella -specific IgA

Dietary cholesterol is metabolized into 25-hydroxycholesterol, which decreases intestinal IgA responses by reducing the expression of sterol-sensing transcription factor SREBP2, thereby preventing IgA plasma cell differentiation.

Maternal-Fetal Immunologic Response to SARS-CoV-2 Infection in a Symptomatic Vulnerable Population: A Prospective Cohort.

Coronavirus disease 2019 (COVID-19) disproportionally affects pregnant women and their newborn; however, little is known about variables that modulate maternal-fetal immune response to infection. We prospectively studied socioeconomic, biologic, and clinical factors affecting humoral immunity in 87 unvaccinated pregnant women hospitalized in Buenos Aires for symptoms consistent with COVID-19. The number of days between symptom onset and childbirth predicted maternal and newborn virus spike protein receptor binding domain (RBD)-specific immunoglobulin G (IgG). These findings suggest newborns may benefit less when mothers deliver soon after COVID-19 infection. Similarly, a longer time between symptom onset and birth predicted higher in utero transfer of maternal IgG and its concentration in cord blood. Older gestational age at birth was associated with lower maternal to cord blood IgG ratio. Of women with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, 87% developed RBD-specific IgA responses in breast milk within 96 hours of childbirth. IgA was not significantly associated with time from infection but correlated with maternal serum IgG and placental transfer. These results demonstrate the combined role of biologic, clinical, and socioeconomic variables associated with maternal RBD-specific antibodies and supports early vaccination strategies for COVID-19 in socioeconomically vulnerable pregnant women. NCT04362956.

Immunoglobulin (Ig)A seropositivity against SARS-CoV-2 in healthcare workers in Israel, 4 April to 13 July 2020: an observational study.

IntroductionThe COVID-19 pandemic has put healthcare workers (HCW) at significant risk. Presence of antibodies can confirm prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.AimThis study investigates the prevalence of IgA and IgG antibodies against SARS-CoV-2 in HCW.MethodsPerformance of IgA and IgG antibody ELISA assays were initially evaluated in positive and negative SARS-CoV-2 serum samples. IgA and IgG antibodies against SARS-CoV-2 were measured in 428 asymptomatic HCW. We assessed the risk of two groups: HCW with high exposure risk outside work (HROW) residing in areas where COVID-19 was endemic (n = 162) and HCW with high exposure risk at work (HRAW) in a COVID-19 intensive care unit (ICU) (n = 97).ResultsSensitivities of 80% and 81.2% and specificities of 97.2% and 98% were observed for IgA and IgG antibodies, respectively. Of the 428 HCW, three were positive for IgG and 27 for IgA. Only 3/27 (11%) IgA-positive HCW had IgG antibodies compared with 50/62 (81%) in a group of previous SARS-CoV-2-PCR-positive individuals. Consecutive samples from IgA-positive HCW demonstrated IgA persistence 18–83 days in 12/20 samples and IgG seroconversion in 1/20 samples. IgA antibodies were present in 8.6% of HROW and 2% of HRAW.ConclusionsSARS-CoV-2 exposure may lead to asymptomatic transient IgA response without IgG seroconversion. The significance of these findings needs further study. Out of work exposure is a possible risk of SARS-CoV-2 infection in HCW and infection in HCW can be controlled if adequate protective equipment is implemented.

Delayed Kinetics of IgG, but Not IgA, Antispike Antibodies in Transplant Recipients following SARS-CoV-2 Infection.

Kidney transplant recipients are at increased risk of severe outcomes during COVID-19. Antibodies against the virus are thought to offer protection, but a thorough characterization of anti-SARS-CoV-2 immune globulin isotypes in kidney transplant recipients following SARS-CoV-2 infection has not been reported. We performed a cross-sectional study of 49 kidney transplant recipients and 42 immunocompetent controls at early (≤14 days) or late (>14 days) time points after documented SARS-CoV-2 infection. Using a validated semiquantitative Luminex-based multiplex assay, we determined the abundances of IgM, IgG, IgG1-4, and IgA antibodies against five distinct viral epitopes. Kidney transplant recipients showed lower levels of total IgG antitrimeric spike (S), S1, S2, and receptor binding domain (RBD) but not nucleocapsid (NC) at early versus late time points after SARS-CoV-2 infection. Early levels of IgG antispike protein epitopes were also lower than in immunocompetent controls. Anti-SARS-CoV-2 antibodies were predominantly IgG1 and IgG3, with modest class switching to IgG2 or IgG4 in either cohort. Later levels of IgG antispike, S1, S2, RBD, and NC did not significantly differ between cohorts. There was no significant difference in the kinetics of either IgM or IgA antispike, S1, RBD, or S2 on the basis of timing after diagnosis or transplant status. Kidney transplant recipients mount early anti-SARS-CoV-2 IgA and IgM responses, whereas IgG responses are delayed compared with immunocompetent individuals. These findings might explain the poor outcomes in transplant recipients with COVID-19. This article contains a podcast at https://www.asn-online.org/media/podcast/JASN/2021_11_23_briggsgriffin112321.mp3.

BNT162b2 vaccine induces divergent B cell responses to SARS-CoV-2 S1 and S2.

The first ever US Food and Drug Administration-approved messenger RNA vaccines are highly protective against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1-3. However, the contribution of each dose to the generation of antibodies against SARS-CoV-2 spike (S) protein and the degree of protection against novel variants warrant further study. Here, we investigated the B cell response to the BNT162b2 vaccine by integrating B cell repertoire analysis with single-cell transcriptomics pre- and post-vaccination. The first vaccine dose elicits a recall response of IgA+ plasmablasts targeting the S subunit S2. Three weeks after the first dose, we observed an influx of minimally mutated IgG+ memory B cells that targeted the receptor binding domain on the S subunit S1 and likely developed from the naive B cell pool. This response was strongly boosted by the second dose and delivers potently neutralizing antibodies against SARS-CoV-2 and several of its variants.

Monomeric IgA Antagonizes IgG-Mediated Enhancement of DENV Infection.

Dengue virus (DENV) is a prevalent human pathogen, infecting approximately 400 million individuals per year and causing symptomatic disease in approximately 100 million. A distinct feature of dengue is the increased risk for severe disease in some individuals with preexisting DENV-specific immunity. One proposed mechanism for this phenomenon is antibody-dependent enhancement (ADE), in which poorly-neutralizing IgG antibodies from a prior infection opsonize DENV to increase infection of Fc gamma receptor-bearing cells. While IgM and IgG are the most commonly studied DENV-reactive antibody isotypes, our group and others have described the induction of DENV-specific serum IgA responses during dengue. We hypothesized that monomeric IgA would be able to neutralize DENV without the possibility of ADE. To test this, we synthesized IgG and IgA versions of two different DENV-reactive monoclonal antibodies. We demonstrate that isotype-switching does not affect the antigen binding and neutralization properties of the two mAbs. We show that DENV-reactive IgG, but not IgA, mediates ADE in Fc gamma receptor-positive K562 cells. Furthermore, we show that IgA potently antagonizes the ADE activity of IgG. These results suggest that levels of DENV-reactive IgA induced by DENV infection might regulate the overall IgG mediated ADE activity of DENV-immune plasma in vivo, and may serve as a predictor of disease risk.

Association of Human Milk Antibody Induction, Persistence, and Neutralizing Capacity With SARS-CoV-2 Infection vs mRNA Vaccination

Long-term effect of parental COVID-19 infection vs vaccination on human milk antibody composition and functional activity remains unclear. To compare temporal IgA and IgG response in human milk and microneutralization activity against SARS-CoV-2 between lactating parents with infection and vaccinated lactating parents out to 90 days after infection or vaccination. Convenience sampling observational cohort (recruited July to December 2020) of lactating parents with infection with human milk samples collected at days 0 (within 14 days of diagnosis), 3, 7, 10, 28, and 90. The observational cohort included vaccinated lactating parents with human milk collected prevaccination, 18 days after the first dose, and 18 and 90 days after the second dose. COVID-19 infection diagnosed by polymerase chain reaction within 14 days of consent or receipt of messenger RNA (mRNA) COVID-19 vaccine (BNT162b2 or mRNA-1273). Human milk anti-SARS-CoV-2 receptor-binding domain IgA and IgG and microneutralization activity against live SARS-CoV-2 virus. Of 77 individuals, 47 (61.0%) were in the infection group (mean [SD] age, 29.9 [4.4] years), and 30 (39.0%) were in the vaccinated group (mean [SD] age, 33.0 [3.4] years; P = .002). The mean (SD) age of infants in the infection and vaccinated group were 3.1 (2.2) months and 7.5 (5.2) months, respectively (P < .001). Infection was associated with a variable human milk IgA and IgG receptor-binding domain-specific antibody response over time that was classified into different temporal patterns: upward trend and level trend (33 of 45 participants [73%]) and low/no response (12 of 45 participants [27%]). Infection was associated with a robust and quick IgA response in human milk that was stable out to 90 days after diagnosis. Vaccination was associated with a more uniform IgG-dominant response with concentrations increasing after each vaccine dose and beginning to decline by 90 days after the second dose. Vaccination was associated with increased human milk IgA after the first dose only (mean [SD] increase, 31.5 [32.6] antibody units). Human milk collected after infection and vaccination exhibited microneutralization activity. Microneutralization activity increased throughout time in the vaccine group only (median [IQR], 2.2 [0] before vaccine vs 10 [4.0] after the first dose; P = .003) but was higher in the infection group (median [IQR], 20 [67] at day 28) vs the vaccination group after the first-dose human milk samples (P = .002). Both IgA and non-IgA (IgG-containing) fractions of human milk from both participants with infection and those who were vaccinated exhibited microneutralization activity against SARS-CoV-2. In this cohort study of a convenience sample of lactating parents, the pattern of IgA and IgG antibodies in human milk differed between COVID-19 infection vs mRNA vaccination out to 90 days. While infection was associated with a highly variable IgA-dominant response and vaccination was associated with an IgG-dominant response, both were associated with having human milk that exhibited neutralization activity against live SARS-CoV-2 virus.

Induction of mucosal and systemic immune responses against the common O78 antigen of an oral inactivated ETEC vaccine in Bangladeshi children and infants

We tested an oral enterotoxigenic Escherichia coli (ETEC) vaccine, ETVAX, consisting of inactivated E. coli overexpressing the most prevalent ETEC colonization factors (CFs) and a toxoid (LCTBA), in Bangladeshi children for capacity to induce mucosal and plasma immune responses against O78 lipopolysaccharide (LPS) expressed on the vaccine strains. The vaccine was given ± double-mutant heat-labile toxin (dmLT) adjuvant. We evaluated the impact of dmLT on anti-O78 LPS immune responses and whether such responses can predict responses against the CFs as a marker for vaccine “take”. Two fractionated doses of ETVAX ± different amounts of dmLT were administered biweekly to groups of children 24–59 (n = 125), 12–23 (n = 97) and 6–11 (n = 158) months of age. Immune responses were evaluated in antibody in lymphocyte supernatants (ALS), fecal extracts and plasma. ALS IgA responses against O78 LPS were induced in 44–49% of the children aged 12–59 months. The magnitudes of the ALS responses were significantly higher in children receiving a half-dose (5 × 1010 bacteria) of ETVAX ± dmLT than in placebo recipients. <10% of the vaccinees aged 6–11 months mounted ALS responses against O78 LPS. However, 49% of the infants developed fecal secretory IgA responses which were significantly more frequent in those receiving a quarter-dose (2.5 × 1010 bacteria) of vaccine + dmLT (62%) compared to a quarter-dose alone (36%). Plasma IgA antibody responses were induced in 80% of older children and 36% of infants. The frequencies of O78 LPS responses in plasma and feces were comparable or higher than against the vaccine CFs in infants. Our findings show that ETVAX induced mucosal and systemic immune responses against O78 LPS in all age groups and that dmLT improved intestinal immune responses among infants. These observations may have implications for more successful use of other oral vaccines based on O antigens in children.

Vitamin A and D Levels Correlate with Outcomes Following Acute Parvovirus B19 Infection in Children with Sickle Cell Disease

Background: Human parvovirus B19, a common childhood infection that typically causes mild disease in healthy individuals, causes life-threatening anemia due to transient red cell aplasia (aplastic crisis) in individuals with sickle cell disease (SCD). This complication often leads to hospitalization and red blood cell transfusions. Limiting the amount of transfusions is imperative to avoid alloimmunization. Strategies to mitigate complications from parvovirus B19 or reduce disease severity in SCD patients are needed. In this prospective observational study, we aim to describe the relationship between vitamin A and D levels and severity of symptoms during parvovirus B19 infection in pediatric patients with SCD.Methods: Twenty pediatric patients with SCD admitted to St. Jude Children's Research Hospital experiencing aplastic crisis and identified as having an active parvovirus B19 infection were under an IRB approved protocol (NCT02261480) and followed prospectively for 120 days post-infection. Acute parvovirus B19 infection was diagnosed by fever and a positive virus-specific IgM ELISA or PCR. Aplastic crisis was defined by worsened anemia without sufficient compensatory reticulocytosis in the setting of an acute parvovirus B19 infection. Virus-specific antibody responses were measured from sera or nasal washes (NW) by ELISA using parvovirus B19 virus-like particles (VLP) as target antigens. Peak serum antibody titers were defined (among measurements from Days 0, 7, 30, and 120). Total erythrocyte transfusion volumes required during the admission were quantified. Hematologic indices and vitamin A and D levels were obtained on Day 0. Vitamin A (retinol) was measured using UPLC and vitamin D (25[OH]D) was measured using a Roche Elecsys Vitamin D ELISA. Statistical analyses were performed with Spearman's rank correlation coefficient or Mann Whitney tests. We scored significance as p<0.05.Results: Twenty patients with different genotypes of SCD requiring hospital admission due to acute parvovirus B19 infection were sequentially enrolled (15 HbSS, 3 HbSC, 1 HbSD, 1 HbSβeta +thalassemia). Thirty-five percent (7/20) of patients were vitamin D deficient (VDD, defined here as <20 ng/mL) while 89% (17/19, one sample was not measured) of patients were marginally or severely vitamin A deficient (defined here as <20 μg/dL) on Day 0. Correlative analyses between vitamin levels and markers of disease severity are shown in figure 1. Lower vitamin D levels trended with lower hemoglobin (r=0.399, p=0.082) and there was a significant inverse correlation between transfused volume and vitamin D levels (r=-0.626, p=0.003, Figure 1B). There was a significant positive correlation of vitamin A levels on Day 0 with hemoglobin concentration (r=0.549, p=0.015, Figure 1D), and an inverse correlation between vitamin A levels and lactate dehydrogenase (r=-0.505, p=0.027, Figure 1F).Children with replete vitamin D levels exhibited significantly better peak virus-specific serum IgG (p=0.037) and IgA (p=0.011) responses (Figure 2A-B). Additionally, vitamin A, which plays an important role in mucosal immunity, correlated positively with virus-specific IgG (r=0.574, p=0.013) and IgA (r=0.480, p=0.044) in nasal passages on Day 30 (Figure 2C-D).Conclusions: In pediatric patients admitted to the hospital for acute parvovirus B19 infection, low vitamin A and D levels were associated with greater disease severity (greater hemolysis, lower hemoglobin concentration, and greater transfusion volumes). Higher vitamin levels were associated with better virus-specific antibody responses. While cause-effect relationships were not discerned in this study, we hypothesize that poor nutritional status contributed to poor outcomes and that efforts to improve nutrition may reduce the severity of parvovirus B19 disease in patients with SCD. [Display omitted] DisclosuresHankins: UpToDate: Consultancy; Vindico Medical Education: Consultancy; Global Blood Therapeutics: Consultancy; Bluebird Bio: Consultancy.

Glycosylation and Serological Reactivity of an Expression-enhanced SARS-CoV-2 Viral Spike Mimetic

Extensive glycosylation of viral glycoproteins is a key feature of the antigenic surface of viruses and yet glycan processing can also be influenced by the manner of their recombinant production. The low yields of the soluble form of the trimeric spike (S) glycoprotein from SARS-CoV-2 has prompted advances in protein engineering that have greatly enhanced the stability and yields of the glycoprotein. The latest expression-enhanced version of the spike incorporates six proline substitutions to stabilize the prefusion conformation (termed SARS-CoV-2 S HexaPro). Although the substitutions greatly enhanced expression whilst not compromising protein structure, the influence of these substitutions on glycan processing has not been explored. Here, we show that the site-specific N-linked glycosylation of the expression-enhanced HexaPro resembles that of an earlier version containing two proline substitutions (2P), and that both capture features of native viral glycosylation. However, there are site-specific differences in glycosylation of HexaPro when compared to 2P. Despite these discrepancies, analysis of the serological reactivity of clinical samples from infected individuals confirmed that both HexaPro and 2P protein are equally able to detect IgG, IgA, and IgM responses in all sera analysed. Moreover, we extend this observation to include an analysis of glycan engineered S protein, whereby all N-linked glycans were converted to oligomannose-type and conclude that serological activity is not impacted by large scale changes in glycosylation. These observations suggest that variations in glycan processing will not impact the serological assessments currently being performed across the globe.