IgA Titers Research Articles

The Screening of Celiac Serology in Pediatric Patients Diagnosed with Type 1 Diabetes Mellitus.

This study aims to detect the rate of celiac antibody test positivity in pediatric patients diagnosed with type 1 diabetes mellitus (T1DM) and determine the characteristics of the patient groups diagnosed with and without celiac disease (CD). This study was conducted retrospectively in Adana City Training and Research Hospital, Pediatric Endocrinology Outpatient Clinic with the patients diagnosed with T1DM between September 17, 2017, and January 1, 2022. The patients were examined by 3 different groups. Group 1: celiac patient group, group 2: serology false positive group, and group 3: serology negative group. The study included 418 patients, 228 (54.5%) males and 190 (45.5%) females. About 6% of the patients (25 patients) were in the celiac patient group, 12.6% (53 patients) in the serology false positive group, and 81.3% (340 patients) in the serology negative group. The age at diagnosis was 10 (7.2-12.9) years in the celiac patient group and 8.8 (6.2-12.00) years in the serology false positive group (P = .559). Among 53 patients in the serology false positive group, spontaneous normalization was observed in 66% (35 patients), and positivity was continued during the test follow-up period in 34% (18 patients). There was no significant difference in terms of sex (P = .101), and HbA1c values at diagnosis (P = .557). Tissue transglutaminase IgA titer was 20× Upper limit of normal (ULN) in the celiac patient group and 2.52× ULN in the serology false positive group. T1DM and CD are both autoimmune diseases concurrently seen together. CD antibody positivity may be observed at the first presentation of T1DM. While the majority of these antibodies become negative during the follow-up, we wanted to highlight the false positive antibody titers and emphasize that these patients should be followed by endocrinologists and gastroenterologists together.

Protective Efficacy of a Mucosal Influenza Vaccine Formulation Based on the Recombinant Nucleoprotein Co-Administered with a TLR2/6 Agonist BPPcysMPEG.

Current influenza vaccines target highly variable surface glycoproteins; thus, mismatches between vaccine strains and circulating strains often diminish vaccine protection. For this reason, there is still a critical need to develop effective influenza vaccines able to protect also against the drift and shift of different variants of influenza viruses. It has been demonstrated that influenza nucleoprotein (NP) is a strong candidate for a universal vaccine, which contributes to providing cross-protection in animal models. In this study, we developed an adjuvanted mucosal vaccine using the recombinant NP (rNP) and the TLR2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxyl-poly-ethylene-glycol (BPPcysMPEG). The vaccine efficacy was compared with that observed following parenteral vaccination of mice with the same formulation. Mice vaccinated with 2 doses of rNP alone or co-administered with BPPcysMPEG by the intranasal (i.n.) route showed enhanced antigen-specific humoral and cellular responses. Moreover, NP-specific humoral immune responses, characterized by significant NP-specific IgG and IgG subclass titers in sera and NP-specific IgA titers in mucosal territories, were remarkably increased in mice vaccinated with the adjuvanted formulation as compared with those of the non-adjuvanted vaccination group. The addition of BPPcysMPEG also improved NP-specific cellular responses in vaccinated mice, characterized by robust lymphoproliferation and mixed Th1/Th2/Th17 immune profiles. Finally, it is notable that the immune responses elicited by the novel formulation administered by the i.n. route were able to confer protection against the influenza H1N1 A/Puerto Rico/8/1934 virus.

VLPs containing stalk domain and ectodomain of matrix protein 2 of influenza induce protection in mice

BackgroundAs a result of antigenic drift, current influenza vaccines provide limited protection against circulating influenza viruses, and vaccines with broad cross protection are urgently needed. Hemagglutinin stalk domain and ectodomain of matrix protein 2 are highly conserved among influenza viruses and have great potential for use as a universal vaccine.MethodsIn this study, we co-expressed the stalk domain and M2e on the surface of cell membranes and generated chimeric and standard virus-like particles of influenza to improve antigen immunogenicity. We subsequently immunized BALB/c mice through intranasal and intramuscular routes.ResultsData obtained demonstrated that vaccination with VLPs elicited high levels of serum-specific IgG (approximately 30-fold higher than that obtained with soluble protein), induced increased ADCC activity to the influenza virus, and enhanced T cell as well as mucosal immune responses. Furthermore, mice immunized by VLP had elevated level of mucosal HA and 4M2e specific IgA titers and cytokine production as compared to mice immunized with soluble protein. Additionally, the VLP-immunized group exhibited long-lasting humoral antibody responses and effectively reduced lung viral titers after the challenge. Compared to the 4M2e-VLP and mHA-VLP groups, the chimeric VLP group experienced cross-protection against the lethal challenge with homologous and heterologous viruses. The stalk domain specific antibody conferred better protection than the 4M2e specific antibody.ConclusionOur findings demonstrated that the chimeric VLPs anchored with the stalk domain and M2e showed efficacy in reducing viral loads after the influenza virus challenge in the mice model. This antibody can be used in humans to broadly protect against a variety of influenza virus subtypes. The chimeric VLPs represent a novel approach to increase antigen immunogenicity and are promising candidates for a universal influenza vaccine.

Epitope Coverage of Anti-SARS-CoV-2 Nucleocapsid IgA and IgG Antibodies Correlates with Protection against Re-Infection by New Variants in Subsequent Waves of the COVID-19 Pandemic.

The COVID-19 pandemic continues to affect individuals across the globe, with some individuals experiencing more severe disease than others. The relatively high frequency of re-infections and breakthrough infections observed with SARS-CoV-2 highlights the importance of extending our understanding of immunity to COVID-19. Here, we aim to shed light on the importance of antibody titres and epitope utilization in protection from re-infection. Health care workers are highly exposed to SARS-CoV-2 and are therefore also more likely to become re-infected. We utilized quantitative, multi-antigen, multi-epitope SARS-CoV-2 protein microarrays to measure IgG and IgA titres against various domains of the nucleocapsid and spike proteins. Potential re-infections in a large, diverse health care worker cohort (N = 300) during the second wave of the pandemic were identified by assessing the IgG anti-N titres before and after the second wave. We assessed epitope coverage and antibody titres between the 'single infection' and 're-infection' groups. Clear differences were observed in the breadth of the anti-N response before the second wave, with the epitope coverage for both IgG (p = 0.019) and IgA (p = 0.015) being significantly increased in those who did not become re-infected compared to those who did. Additionally, the IgG anti-N (p = 0.004) and anti-S titres (p = 0.018) were significantly higher in those not re-infected. These results highlight the importance of the breadth of elicited antibody epitope coverage following natural infection in protection from re-infection and disease in the COVID-19 pandemic.

Preparation of chitosan nanoparticle containing recombinant CD44v antigen and evaluation of its immunization capacity against breast cancer in BALB/c mice

Objective(s)Breast tumors show heterogeneity containing cancer stem cells as a small subpopulation of a tumor mass. CD44 as a cancer stem cells antigen is abnormally expressed by carcinomas of epithelial origin. Also, overexpression of CD44 variable isoforms (CD44v) is associated with malignancy in breast cancer. In the present research, our objective was to evaluate the immunogenicity of prepared nanoparticles containing a novel recombinant CD44v (rCD44v) protein in the mouse model.Materials and methodsCD44 gene was expressed in E. coli BL21 DE3 using the pET28a-CD44 vector. The expressed rCD44v protein was purified, encapsulated into the chitosan nanoparticles, and administered to BALB/c mice. ELISA was used to evaluate the immunoglobulin levels of immunized animals. For challenge experiment, 2 × 106 4T1-CD44 tumor cells were injected subcutaneously in mice, and tumor size, necrosis, and metastases were measured. Finally, cell proliferation assay, cytokines assay, and neutralization assay of the mouse anti-rCD44v on the human breast cancer cell line were examined.ResultsThe measured size of chitosan-rCD44v nanoparticles was 146.5 nm. Recombinant CD44v encapsulated by chitosan nanoparticles increases immunological responses via the adjuvant nature of chitosan nanoparticles. In the immunized mice, IgG and IgA titers were significantly increased. Tumor growth in injection and nano-injection test groups compared with the mice control groups displayed a significant reduction (P < 0.05). A high amount of splenocytes secreting IFNγ and IL-17 was seen in immunized mice with rCD44v (P < 0.05). Furthermore, a smaller size of lung metastases compared to the control mice groups was detected.ConclusionThe encapsulated rCD44v within the chitosan nanoparticles induced a significant immune response in mice and can establish significant protection against breast cancer. Therefore, it can be considered a vaccine candidate for breast cancer therapeutic modalities.

Longitudinal IgA and IgG Response, and ACE2 Binding Blockade, to Full-Length SARS-CoV-2 Spike Protein Variants in a Population of Black PLWH Vaccinated with ChAdOx1 nCoV-19.

Vaccines against SARS-CoV-2 have been pivotal in overcoming the COVID-19 pandemic yet understanding the subsequent outcomes and immunological effects remain crucial, especially for at-risk groups e.g., people living with human immunodeficiency virus (HIV) (PLWH). In this study we report the longitudinal IgA and IgG antibody titers, as well as antibody-mediated angiotensin converting enzyme 2 (ACE2) binding blockade, against the SARS-CoV-2 spike (S) proteins after 1 and 2 doses of the ChAdOx1 nCoV-19 vaccine in a population of Black PLWH. Here, we report that PLWH (N = 103) did not produce an anti-S IgA response after infection or vaccination, however, anti-S IgG was detected in response to vaccination and infection, with the highest level detected for infected vaccinated participants. The anti-IgG and ACE2 blockade assays revealed that both vaccination and infection resulted in IgG production, however, only vaccination resulted in a moderate increase in ACE2 binding blockade to the ancestral S protein. Vaccination with a previous infection results in the greatest anti-S IgG and ACE2 blockade for the ancestral S protein. In conclusion, PLWH produce an anti-S IgG response to the ChAdOx1 nCoV-19 vaccine and/or infection, and ChAdOx1 nCoV-19 vaccination with a previous infection produced more neutralizing antibodies than vaccination alone.

Mucosal immune response after the booster dose of the BNT162b2 COVID-19 vaccine.

To date, only a few studies reported data regarding the development of mucosal immune response after the BNT162b2-booster vaccination. Samples of both serum and saliva of 50 healthcare workers were collected at the day of the booster dose (T3) and after two weeks (T4). Anti-S1-protein IgG and IgA antibody titres and the neutralizing antibodies against the Wuhan wild-type Receptor-Binding Domain in both serum and saliva were measured by quantitative and competitive ELISA, respectively. Data were compared with those recorded after the primary vaccination cycle (T2). Neutralizing antibodies against the variants of concern were measured in those individuals with anti-Wuhan neutralizing antibodies in their saliva. After eight months from the second dose, IgG decreased in both serum (T2GMC: 23,838.5ng/ml; T3GMC: 1473.8ng/ml) and saliva (T2GMC: 12.9ng/ml; T3GMC: 0.3ng/ml). Consistently, serum IgA decreased (T2GMC: 48.6ng/ml; T3GMC: 6.4ng/ml); however, salivary IgA showed a different behaviour and increased (T2GMC: 0.06ng/ml; T3GMC: 0.41ng/ml), indicating a delayed activation of mucosal immunity. The booster elicited higher titres of both IgG and IgA when compared with the primary cycle, in both serum (IgG T4GMC: 98,493.9ng/ml; IgA T4GMC: 187.5ng/ml) and saliva (IgG T4GMC: 21.9ng/ml; IgA T4GMC: 0.65ng/ml). Moreover, the booster re-established the neutralizing activity in the serum of all individuals, not only against the Wuhan wild-type antigen (N=50; INH: 91.6%) but also against the variants (Delta INH: 91.3%; Delta Plus INH: 89.8%; Omicron BA.1 INH: 85.1%). By contrast, the salivary neutralizing activity was high against the Wuhan antigen in 72% of individuals (N=36, INH: 62.2%), but decreased against the variants, especially against the Omicron BA.1 variant (Delta N=27, INH: 43.1%; Delta Plus N=24, INH: 35.2%; Omicron BA.1N=4; INH: 4.7%). This was suggestive for a different behaviour of systemic immunity observed in serum with respect to mucosal immunity described in saliva (Wald chi-square test, 3 df of interaction between variants and sample type=308.2, p<0.0001). The BNT162b2-booster vaccination elicits a strong systemic immune response but fails in activating an effective mucosal immunity against the Omicron BA.1 variant. This work was funded by the Department of Medicine and Surgery, University of Insubria, and supported by Fondazione Umberto Veronesi (COVID-19 Insieme per la ricerca di tutti, 2020), Italy.

WHICH IS IMPORTANT? GLUTEN FREE DIET OR INFLAMMATORY BOWEL DISEASE MEDICATIONS IN SUBJECTS WITH AN OVERLAP WITH CELIAC DISEASE AND INFLAMMATORY BOWEL DISEASE: OBSERVATIONS FROM A CASE SERIES AT A LARGE ACADEMIC CENTER

Abstract INTRODUCTION Celiac disease (CeD) and inflammatory bowel disease (IBD) are autoimmune diseases of the bowel with similar clinical symptoms. This study aims to characterize the clinical course of both diseases in subjects with overlap of CeD and IBD (CeD+IBD) on a gluten free diet (GFD) and IBD medications. METHODS We conducted a retrospective study of demographic and serological characteristics in subjects with CeD+ IBD at a large academic center using appropriate International Classification of Disease (ICD)-10 codes from 2017 to June 2022. RESULTS 36 subjects had IBD +CeD. 96% of subjects were Caucasian and 67% were female. The diagnosis of CeD post-dated the diagnosis of IBD by 3.6 years. Ulcerative colitis (UC) and Crohn’s disease (CD) occurred equally in IBD +CeD. About 75% of subjects had L2+L3, B1 phenotype per Montreal classification. 44% of UC +CeD subjects had pancolitis with 72% of them being classified as S0 (never severe) per Paris classification. 83% of subjects initiated a self-taught GFD. Only 17% of subjects sough advice from a celiac dietician. Mean duration at which GFD was initiated was 0.7 yrs from the initial diagnosis of CeD. Only 1/36 subjects followed a strict GFD during 6.5 years of follow up. The second and third assessment of GFD happened after a mean of 3.7 yrs and 5.2 yrs from the time of initiation of a GFD with only 1/36 subjects adhering to a strict GFD at these time points. Serum tTG IgA levels were tested on average at 4, 91, 154, 186 and 192 weeks from the diagnosis of CeD, with a consistent drop in mean tTG IgA titers to 45, 33, 19, 25 and 4 respectively. Only 1 subject got tTG IgA measurements over the next 250 weeks of follow up. Over a 10.1 year follow up since the diagnosis of IBD, descending proportions of subjects needing IBD medications were as follows: topical 5-ASA (aminosalicyates) (19/36)&amp;gt;oral steroids (18/36)&amp;gt; anti-TNF (tumor necrosis factor) (18/36)&amp;gt; anti-integrins (12/36)&amp;gt; azathioprine (10/36)&amp;gt; anti-IL12/23 (8/36)&amp;gt; topical 5-ASA (4/36)&amp;gt; MTX (methotrexate) (5/36)&amp;gt; topical steroids at 2/36. Mean time to initiation of IBD medications (in weeks) after GFD initiation as a reference point were in an order of oral 5-ASA (-67) &amp;lt; anti-TNF (-39) &amp;lt; AZA (-14) &amp;lt;MTX (-6) &amp;lt; topical 5-ASA (-2) &amp;lt;oral steroids (82) &amp;lt;anti-integrins (124) &amp;lt;anti-IL 12/23 (137) CONCLUSIONS Majority of subjects depended on a self-educated GFD. A small proportion of CeD +IBD subjects were assessed per clinical guidelines with respect to adherence to a GFD, serology. At least 50% of subjects with CeD+IBD needed escalation of IBD treatment to biologics and/or steroid rescue over 10 years despite initiation of GFD (albeit suboptimal). We need to further substantiate these findings with exclusive IBD and celiac control groups. We suggest establishing CeD centers at tertiary hospitals for adequate clinical guideline adherence.

The role of serology in the diagnosis of coeliac disease.

Serology has significantly revolutionized the knowledge of celiac disease (CD), leading to the identification of unsuspected patients in at-risk CD groups, thereby increasing the number of CD diagnoses compared to the pre-screening era. Several markers for CD with a progressive diagnostic accuracy have been identified over the years, but only three of them, i.e. anti-tissue transglutaminase (anti-tTG), anti-endomysial (EmA) and anti-deamidated gliadin antibodies (DGP) are currently assessed in the daily clinical practice. A thorough review of the literature identified 44 original studies published between 1998 to 2022 for a total of 5098 pediatric and adult CD patients (without selective IgA deficiency) and 11930 disease controls. The results highlighted that anti-tTG IgA exhibited a higher sensitivity for CD (93.4%) than EmA IgA (92.8%), DGP IgG (81.8%) and DGP IgA (83.8%). The specificity of EmA IgA (99%) resulted to be higher than those of anti-tTG IgA (95.8%), DGP IgG (96.4%) and DGP IgA (92.1%). In patients with selective IgA deficiency, a condition closely related to CD, serological screening should include one of the three antibodies of IgG class, since anti-tTG, DGP and EmA have a very similar diagnostic accuracy in this clinical setting. According to age, there are two main diagnostic strategies for CD detection. In children, the revised ESPGHAN 2020 guidelines established that CD could be diagnosed in both symptomatic and asymptomatic children by high anti-tTG IgA titers (>10 times the cut-off) and EmA positivity with no need to obtain duodenal biopsy and HLA typing. In adult patients, although high tTG IgA titers (confirmed by EmA IgA positivity) correlate with villous atrophy, an intestinal biopsy is still considered mandatory for confirming CD diagnosis. Currently, a case finding approach in at-risk groups is preferred to mass screening for CD detection.

Microbial infections as potential risk factors for lung cancer: Investigating the role of human papillomavirus and chlamydia pneumoniae.

Lung cancer is the leading cause of cancer morbidity and mortality worldwide. Apart from tobacco smoke and dietary factors, microbial infections have been reported as the third leading cause of cancers globally. Deciphering the association between microbiome and lung cancer will provide potential biomarkers and novel insight in lung cancer progression. In this current study, we performed a meta-analysis to decipher the possible association between C. pneumoniae and human papillomavirus (HPV) and the risk of lung cancer. Literature search was conducted in most English and Chinese databases. Data were analyzed using CMA v.3.0 and RevMan v.5.3 software (Cochrane-Mantel-Haenszel method) by random-effects (DerSimonian and Laird) model. The overall pooled estimates for HPV studies revealed that HPV infections in patients with lung cancer were significantly higher than those in the control group (OR = 2.33, 95% CI = 1.57-3.37, p < 0.001). Base on subgroup analysis, HPV infection rate was significantly higher in Asians (OR = 6.38, 95% CI = 2.33-17.46, p < 0.001), in tissues (OR = 5.04, 95% CI = 2.27-11.19, p < 0.001) and blood samples (OR = 1.40, 95% CI = 1.02-1.93, p = 0.04) of lung cancer patients but non-significantly lower in males (OR = 0.84, 95% CI = 0.57-1.22, p =0.35) and among lung cancer patients at clinical stage I-II (OR = 0.95, 95% CI = 0.61-1.49, p = 0.82). The overall pooled estimates from C. pneumoniae studies revealed that C. pneumoniae infection is a risk factor among lung cancer patients who are IgA seropositive (OR = 1.88, 95% CI = 1.30-2.70, p < 0.001) and IgG seropositive (OR = 1.50, 95% CI = 1.10-2.04, p = 0.010). All seronegative IgA (OR = 0.69, 95% CI = 0.42-1.16, p = 0.16) and IgG (OR = 0.66, 95% CI = 0.42-105, p = 0.08) titers are not associative risk factors to lung cancer. Immunoglobulin (IgA) and IgG seropositive titers of C. pneumoniae and lungs infected with HPV types 16 and 18 are potential risk factors associated with lung cancer.

SARS-CoV-2-specific nasal IgA wanes 9 months after hospitalisation with COVID-19 and is not induced by subsequent vaccination.

Most studies of immunity to SARS-CoV-2 focus on circulating antibody, giving limited insights into mucosal defences that prevent viral replication and onward transmission. We studied nasal and plasma antibody responses one year after hospitalisation for COVID-19, including a period when SARS-CoV-2 vaccination was introduced. In this follow up study, plasma and nasosorption samples were prospectively collected from 446 adults hospitalised for COVID-19 between February 2020 and March 2021 via the ISARIC4C and PHOSP-COVID consortia. IgA and IgG responses to NP and S of ancestral SARS-CoV-2, Delta and Omicron (BA.1) variants were measured by electrochemiluminescence and compared with plasma neutralisation data. Strong and consistent nasal anti-NP and anti-S IgA responses were demonstrated, which remained elevated for nine months (p<0.0001). Nasal and plasma anti-S IgG remained elevated for at least 12 months (p<0.0001) with plasma neutralising titres that were raised against all variants compared to controls (p<0.0001). Of 323 with complete data, 307 were vaccinated between 6 and 12 months; coinciding with rises in nasal and plasma IgA and IgG anti-S titres for all SARS-CoV-2 variants, although the change in nasal IgA was minimal (1.46-fold change after 10 months, p=0.011) and the median remained below the positive threshold determined by pre-pandemic controls. Samples 12 months after admission showed no association between nasal IgA and plasma IgG anti-S responses (R=0.05, p=0.18), indicating that nasal IgA responses are distinct from those in plasma and minimally boosted by vaccination. The decline in nasal IgA responses 9 months after infection and minimal impact of subsequent vaccination may explain the lack of long-lasting nasal defence against reinfection and the limited effects of vaccination on transmission. These findings highlight the need to develop vaccines that enhance nasal immunity. This study has been supported by ISARIC4C and PHOSP-COVID consortia. ISARIC4C is supported by grants from the National Institute for Health and Care Research and the Medical Research Council. Liverpool Experimental Cancer Medicine Centre provided infrastructure support for this research. The PHOSP-COVD study is jointly funded by UK Research and Innovation and National Institute of Health and Care Research. The funders were not involved in the study design, interpretation of data or the writing of this manuscript.

Probiotic Debaryomyces hansenii CBS 8339 yeast enhanced immune responses in mice.

This study aimed to examine the effect of Debaryomyces hansenii CBS 8339 on innate immune responses in mice. Thirty BALB/c mice were randomly treated with phosphate buffered saline (PBS) (control) and two D. hansenii (Dh) doses: Dh 10ˆ6 CFU (colony forming units) and Dh 10ˆ8 CFU daily for 15days. Spleen, blood, and gut samples were taken on days7 and 15. Mouse splenocytes were isolated and challenged with Escherichia coli. Immunological assays and immune-related gene expressions were performed. Serum was obtained from blood for total IgA and IgG antibody titer determination. Gut samples were taken for yeast colonization assessment. Phagocytosis, respiratory burst activity, and nitric oxide production in mice were mainly enhanced (p < 0.05) upon 7days of D. hansenii intake at a concentration of 10ˆ8 CFU before and after bacterial challenge. Moreover, oral D. hansenii in mice upregulated (p < 0.05) gene expression of pro-inflammatory cytokines (INF-γ, IL-6 and IL-1β) before or after E. coli challenge on day7 but downregulated (p < 0.05) on day15. Furthermore, total serum IgG and IgA titers were higher (p < 0.05) in Dh 10ˆ8 CFU at days7 and 15, and only at day7, respectively, than that in the other dose and control groups. Finally, D. hansenii was detected in the gut of mice that received the treatments, suggesting that yeast survived gastrointestinal transit. Altogether, a short period (7days) of D. hansenii CBS 8339 oral delivery improved immune innate response on mice.

Characterization of Systemic and Mucosal Humoral Immune Responses to an Adjuvanted Intranasal SARS-CoV-2 Protein Subunit Vaccine Candidate in Mice.

Continuous viral evolution of SARS-CoV-2 has resulted in variants capable of immune evasion, vaccine breakthrough infections and increased transmissibility. New vaccines that invoke mucosal immunity may provide a solution to reducing virus transmission. Here, we evaluated the immunogenicity of intranasally administered subunit protein vaccines composed of a stabilized SARS-CoV-2 spike trimer or the receptor binding domain (RBD) adjuvanted with either cholera toxin (CT) or an archaeal lipid mucosal adjuvant (AMVAD). We show robust induction of immunoglobulin (Ig) G and IgA responses in plasma, nasal wash and bronchoalveolar lavage in mice only when adjuvant is used in the vaccine formulation. While the AMVAD adjuvant was more effective at inducing systemic antibodies against the RBD antigen than CT, CT was generally more effective at inducing overall higher IgA and IgG titers against the spike antigen in both systemic and mucosal compartments. Furthermore, vaccination with adjuvanted spike led to superior mucosal IgA responses than with the RBD antigen and produced broadly targeting neutralizing plasma antibodies against ancestral, Delta and Omicron variants in vitro; whereas adjuvanted RBD elicited a narrower antibody response with neutralizing activity only against ancestral and Delta variants. Our study demonstrates that intranasal administration of an adjuvanted protein subunit vaccine in immunologically naïve mice induced both systemic and mucosal neutralizing antibody responses that were most effective at neutralizing SARS-CoV-2 variants when the trimeric spike was used as an antigen compared to RBD.

The microRNA processing subunit DGCR8 is required for a T cell-dependent germinal center response.

We have previously shown that the microRNA (miRNA) processor complex consisting of the RNAse Drosha and the DiGeorge Critical Region (DGCR) 8 protein is essential for B cell maturation. To determine whether miRNA processing is required to initiate T cell-mediated antibody responses, we deleted DGCR8 in maturing B2 cells by crossing a mouse with loxP-flanked DGCR8 alleles with a CD23-Cre mouse. As expected, non-immunized mice showed reduced numbers of mature B2 cells and IgG-secreting cells and diminished serum IgG titers. In accordance, germinal centers and antigen-specific IgG-secreting cells were absent in mice immunized with T-dependent antigens. Therefore, DGCR8 is required to mount an efficient T-dependent antibody response. However, DGCR8 deletion in B1 cells was incomplete, resulting in unaltered B1 cell numbers and normal IgM and IgA titers in DGCR8-knock-out mice. Therefore, this mouse model could be used to analyze B1 responses in the absence of functional B2 cells.

505. Mucosal and Systemic Humoral Immunity Differences between Sexes during Influenza Vaccination and Viral Challenge

Abstract Background In previous influenza challenge studies we observed that the incidence of influenza symptoms was higher in women than in men, and that women experience more symptoms overall. Women also demonstrated lower neuraminidase (NA) inhibition (NAI) antibody titers than men after challenge and regression analysis suggested that NAI titers predict clinical outcomes. We sought to evaluate this using data from a more recent challenge study with more immunologically varied volunteers, while also assessing mucosal immunity. Methods We obtained mucosal and serum samples from healthy adults. Half underwent intramuscular quadrivalent influenza vaccination followed by influenza A (H1N1) intranasal challenge while others just challenge alone. Antibody titers and clinical outcomes between sexes were compared. Results No significant differences in shedding or symptom outcomes were observed between sexes. Similarly, no differences in systemic titers against NA or hemagglutinin (HA) were seen. Serum total IgG and IgA were also similar between sexes. Prior to vaccination, mucosal IgA titers were significantly lower against HA stalk and NA in women, but these differences disappeared after vaccination. In the unvaccinated group, lower nasal IgA titers were also seen in women pre-challenge. At 7 days post-challenge, differences in mucosal titers between sexes disappeared. However, at 56 days post-challenge, mucosal IgA titers against HA, HA stalk, and NA were all significantly lower in women. Conclusion In contrast to our previous findings, no differences in clinical outcomes or systemic NAI titers were observed between men and women, possibly due to differences in underlying immunity between study populations or the lower sample size in this study. However, mucosal differences were noted with women having lower mucosal titers than men against HA stalk and NA at baseline. While influenza vaccination and challenge eliminated differences in the short term, they reappeared 8 weeks after challenge, with all nasal IgA antibody titers (HA, HA stalk, and NA) lower in women. Further studies are necessary to understand the differences in influenza disease and immunity between sexes. Disclosures All Authors: No reported disclosures.

2136. Development of Recombinant Rotavirus Vaccine Carrying Herpes Simplex Virus 2 Gene Based on Reverse Genetics Technology

Abstract Background Herpes simplex virus type 2 (HSV-2) can cause genital herpes, which reduces the patient's QOL associated with pain. In addition, genital herpes can increase the risk of human immunodeficiency virus infection. Vaccine development has been attempted for many years, but it has not yet been put into practical use. Meanwhile, rotavirus (RV) is one of the major viral causes of severe gastroenteritis in infants and young children worldwide. Currently, two live attenuated vaccines are widely used to prevent severe RV infection in children. We have developed entirely plasmid-based reverse genetics systems of RV, which allowed the generation of recombinant reassortant RV containing a foreign gene. In this study, we sought to develop simian RV (SA11), in which HSV-2 glycoprotein D (gD2) gene was inserted by reverse genetics system, as a herpes vaccine, and evaluated its immunogenicity in mice. Construction of HSV-2 protein expressing rotavirus as a vaccine inducing mucosal immunity Methods Artificially synthesized gD2 DNA fragment was inserted into T7 expression NSP1 plasmids (pT7/NSP1-gD2). Using the plasmid, recombinant SA11-gD2 virus (rSA11-gD2) was generated. gD2 protein expression in infected MA104 cells was verified by Western blotting. rSA11-gD2 was orally inoculated into BALB/c suckling mice twice two weeks apart and into eight-week-old mice three times apart four weeks. Diarrhea score was evaluated for seven days after first inoculation in suckling mice. Serum IgG and IgA titers against RV and gD2 were measured by ELISA. Results Insertion of the gD2 gene and synthesis of gD2 protein were demonstrated by sequence analysis and Wester blotting analysis, respectively. Diarrhea occurred after the first inoculation of rSA11-gD2 in suckling mice. Although IgG and IgA antibodies against RV were induced, gD2 antibody was not detected in convalescent sera in the suckling mice. Meanwhile, in the eight-week-old mice inoculated with three times of rSA11-gD2, significant increases in not only IgG and IgA against RV but also IgG against gD2 were demonstrated. Conclusion Simian RV containing gD2 gene, which was developed by the reverse genetics, induced IgG antibody against gD2 in orally inoculated mice. This strategy can be used to develop genital herpes vaccine. Disclosures All Authors: No reported disclosures.

2347. Blood Transcriptional Correlates of Vaccine-Induced Protection Against TB in Macaques

Abstract Background Identifying correlates of vaccine-induced protection against tuberculosis (TB) is essential for improved vaccine development and evaluation. Prior efforts have focused on antigen-specific CD4 T cell responses measured weeks following vacination, which have repeatedly failed to correlate with protection. Methods Here, we analyzed the blood transcriptional response to vaccination in rhesus macaques vaccinated intravenously (IV) with high- (N=16) or low-dose (N=18) BCG that exhibited varying protection against Mtb challenge six months later. We identified correlates of protection in high-dose recipients and validated in low-dose recipients and in an independent cohort of BCG-vaccinated macaques. We also compared the blood transcriptional response to adaptive responses in the bronchoalveolar lavage (BAL). Results We identified and independently validated seven modules of 2,499 genes induced two days, two weeks, four weeks, and/or twelve weeks post-vaccination. Adaptive modules were associated with protection against challenge in low-dose recipients but not high-dose recipients. In contrast, an innate module (Module 1) on day 2 post-vaccination was associated with protection outcomes eight months later in both high- and low-dose recipients, as well as in an independent cohort of macaques. Module 1 scores at day 2 post-vaccination were also highly correlated with BAL adaptive responses, including Mtb-specific IgA and IgG titers at week 4, log T cell (CD4+, CD8+, MAIT, Vg9) counts at week 4, and antigen-specific CD4+ T cell responses (IFNg, IL2, IL21, TNF) at week 8 (r &amp;gt;0.7, p&amp;lt; 0.0001). None of these lung adaptive markers could reliably predict protection in both high- and low-dose recipients. However, module 1 scores at day 2 post-vaccination accurately predicted protection following Mtb challenge six months later (high-dose recipients, AUC=0.8; low-dose recipients, AUC=0.99). The early innate response in peripheral blood correlates with outcomes post-challenge 8 months later. Correlation between module 1 (innate) scores on day 2 post-vaccination and total thoracic colony forming units (CFU) and number of granulomas following challenge eight months later in high- and low-dose recipients (A-B) and in an independent cohort of macaques vaccinated with BCG through different routes (C-D). AE, aerosol; HD-ID, high dose intradermal; ID, intradermal; ID/AE, intradermal and aerosol; IV, intravenous. The early innate response in peripheral blood is highly correlated with lung CD4 T cell responses 8 weeks post-vaccination. Correlation between module 1 (innate) scores at day 2 post-vaccination and cytokine expression by CD4 T cells in bronchoalveolar lavage (BAL) upon whole cell lysate restimulation. Anyg2T17, any of IFNg, IL2, TNF, or IL17. Conclusion These results suggest that adaptive responses are necessary but not sufficient for BCG-induced protection. Instead, the early innate response in peripheral blood may be a more reliable and generalizable correlate of BCG-induced protection. Disclosures Purvesh Khatri, PhD, Cepheid, Inc.: Advisor/Consultant|Inflammatix, Inc.: Advisor/Consultant|Inflammatix, Inc.: Inflammatix is in negotiations to license the 9-gene signature discussed here.|Inflammatix, Inc.: Stocks/Bonds.

A split influenza vaccine formulated with a combination adjuvant composed of alpha-d-glucan nanoparticles and a STING agonist elicits cross-protective immunity in pigs

BackgroundSwine influenza A viruses (SwIAVs) pose an economic and pandemic threat, and development of novel effective vaccines is of critical significance. We evaluated the performance of split swine influenza A virus (SwIAV) H1N2 antigens with a plant-derived nanoparticle adjuvant alone (Nano-11) [Nano11-SwIAV] or in combination with the synthetic stimulator of interferon genes (STING) agonist ADU-S100 (NanoS100-SwIAV). Specific pathogen free (SPF) pigs were vaccinated twice via intramuscular (IM) or intradermal (ID) routes and challenged with a virulent heterologous SwIAV H1N1-OH7 virus.ResultsAnimals vaccinated IM or ID with NanoS100-SwIAV had significantly increased cross-reactive IgG and IgA titers in serum, nasal secretion and bronchoalveolar lavage fluid at day post challenge 6 (DPC6). Furthermore, NanoS100-SwIAV ID vaccinates, even at half the vaccine dose compared to their IM vaccinated counterparts, had significantly increased frequencies of CXCL10+ myeloid cells in the tracheobronchial lymph nodes (TBLN), and IFNγ+ effector memory T-helper/memory cells, IL-17A+ total T-helper/memory cells, central and effector memory T-helper/memory cells, IL-17A+ total cytotoxic T-lymphocytes (CTLs), and early effector CTLs in blood compared with the Nano11-SwIAV group demonstrating a potential dose-sparing effect and induction of a strong IL-17A+ T-helper/memory (Th17) response in the periphery. However, the frequencies of IFNγ+ late effector CTLs and effector memory T-helper/memory cells, IL-17A+ total CTLs, late effector CTLs, and CXCL10+ myeloid cells in blood, as well as lung CXCL10+ plasmacytoid dendritic cells were increased in NanoS100-SwIAV IM vaccinated pigs. Increased expression of IL-4 and IL-6 mRNA was observed in TBLN of Nano-11 based IM vaccinates following challenge. Furthermore, the challenge virus load in the lungs and nasal passage was undetectable in NanoS100-SwIAV IM vaccinates by DPC6 along with reduced macroscopic lung lesions and significantly higher virus neutralization titers in lungs at DPC6. However, NanoS100-SwIAV ID vaccinates exhibited significant reduction of challenge virus titers in nasal passages and a remarkable reduction of challenge virus in lungs.ConclusionsDespite vast genetic difference (77% HA gene identity) between the H1N2 and H1N1 SwIAV, the NanoS100 adjuvanted vaccine elicited cross protective cell mediated immune responses, suggesting the potential role of this combination adjuvant in inducing cross-protective immunity in pigs.Graphical

Mucosal and Systemic Responses to Severe Acute Respiratory Syndrome Coronavirus 2 Vaccination Determined by Severity of Primary Infection.

With much of the world infected with or vaccinated against severe acute respiratory syndrome coronavirus 2 (commonly abbreviated SARS-CoV-2; abbreviated here SARS2), understanding the immune responses to the SARS2 spike (S) protein in different situations is crucial to controlling the pandemic. We studied the clinical, systemic, mucosal, and cellular responses to two doses of SARS2 mRNA vaccines in 62 individuals with and without prior SARS2 infection that were divided into three groups based on antibody serostatus prior to vaccination and/or degree of disease symptoms among those with prior SARS2 infection: antibody negative (naive), low symptomatic, and symptomatic. Antibody negative were subjects who were antibody negative (i.e., those with no prior infection). Low symptomatic subjects were those who were antibody negative and had minimal or no symptoms at time of SARS2 infection. Symptomatic subjects were those who were antibody positive and symptomatic at time of SARS2 infection. All three groups were then studied when they received their SARS2 mRNA vaccines. In the previously SARS2-infected (based on antibody test) low symptomatic and symptomatic groups, reactogenic symptoms related to a recall response were elicited after the first vaccination. Anti-S trimer IgA and IgG titers, and neutralizing antibody titers, peaked after the 1st vaccination in the previously SARS2-infected groups and were significantly higher than for the SARS2 antibody-negative group in the plasma and nasal samples at most time points. Nasal and plasma IgA antibody responses were significantly higher in the low symptomatic group than in the symptomatic group at most time points. After the first vaccination, differences in cellular immunity were not evident between groups, but the activation-induced cell marker (AIM+) CD4+ cell response correlated with durability of IgG humoral immunity against the SARS2 S protein. In those SARS2-infected subjects, severity of infection dictated plasma and nasal IgA responses in primary infection as well as response to vaccination (peak responses and durability), which could have implications for continued protection against reinfection. Lingering differences between the SARS2-infected and SARS2-naive up to 10 months postvaccination could explain the decreased reinfection rates in the SARS2-infected vaccinees recently reported and suggests that additional strategies (such as boosting of the SARS2-naive vaccinees) are needed to narrow the differences observed between these groups. IMPORTANCE This study on SARS2 vaccination in those with and without previous exposure to the virus demonstrates that severity of infection dictates IgA responses in primary infection as well as response to vaccination (peak responses and durability), which could have implications for continued protection against reinfection.

PSAT118 Elevated Serum IgA at Onset of Type 1 Diabetes in Children

Abstract IgA is the most produced antibody in the body. There are two major forms of IgA, monomeric serum IgA and dimeric mucosal (secretory) IgA. During infection or inflammation, IgA binding to its antigen induces crosslinking of FcaRI, which activates immune effector cells to carry out their functions and can thus induce pro-inflammatory responses, which is hypothesized to occur when IgA titers are high. (1,2) Elevated serum IgA has been associated with autoimmune diseases (3) including type 1 diabetes (T1D) but its role in disease pathogenesis is not well known. To address this gap in knowledge, we aimed to test the hypothesis that at diagnosis of T1D, children and adolescents with elevated serum IgA have different demographic, clinical and laboratory characteristics than those with normal serum IgA. We analyzed 612 children 9.7 [4.2] (mean [SD]) years old (59% Non-Hispanic White, 20% Hispanic, 16% Black, 5% Other) with new onset T1D diagnosed between 1/2008-2/2012 at a large, academic pediatric hospital. Serum IgA values above age-adjusted normal ranges were defined as elevated serum IgA. Patients with low IgA were excluded because of known association between IgA deficiency and autoimmune disease. Demographic and clinical variables were compared between youth with and without elevated serum IgA. Univariable and multivariable regression models were used to compare baseline characteristics that were associated with elevated serum IgA. A significance level of 0.05 was used. We found that elevated serum IgA was present in 21% (128/612) of the children at T1D onset. By multivariate analysis, compared to children with normal IgA, those with elevated IgA were more likely to be of Hispanic ethnicity (Odds Ratio (OR)=3.27, 95% Confidence Interval (CI)=2.00, 5.35) and had a higher hemoglobin A1c (A1c) (OR=1.13, 95% CI=1.01, 1.26). They also had higher odds of having insulin autoantibody (IAA) positivity at diagnosis (OR=1.65, 95% CI=1.10, 2.45) and lower odds of GAD autoantibody (GADA) positivity (OR= 0.57, 95% CI=0.36, 0.93) (all, p&amp;lt;0.05), while there were no significant associations with age, glucose level or presence of DKA. In sum, at diagnosis of T1D, 21% of youth have elevated serum IgA and this was associated with Hispanic ethnicity, higher A1C and positivity for IAA. Our findings suggest that elevated serum IgA may help identify youth with T1D, islet autoimmunity and insulin deficiency. Further studies are warranted to investigate potential pathogenic correlates and persistence over time. This knowledge may lead to new targeted strategies to preserve or improve beta-cell function in individuals with clinical or pre-clinical T1D. References (1) Hansen IS et al. Cell Mol Life Sci. 2019 Mar;76(6): 1041-1055. PMID: 30498997 (2) Leong KW et al. DNA Cell Biol. 2014 Dec;33(12): 823-9. PMID: 25188736 (3) Kerr MA. Biochem J. 1990 Oct 15;271(2): 285-96. PMID: 2241915 Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.