High Ab Levels Research Articles

Anti-Lipoprotein Lipase Antibody as a Useful Marker for Plaque Vulnerability in Patients with Stable Angina.

Identifying patients with vulnerable plaque who have poor prognosis among those with coronary artery disease (CAD) is crucial to deciding future therapeutic interventions. We previously reported that male CAD patients with low anti-apolipoprotein B-100 autoantibody (anti-apoB-100 Ab) levels were at an increased risk of developing unstable plaque lesions. This study focused on the autoantibodies against lipoprotein lipase (LPL), a key enzyme in triglyceride metabolism, which is another risk factor for atherosclerosis, and investigated their association with plaque characteristics. We measured serum anti-LPL Ab levels using a homemade enzyme-linked immunosorbent assay in 80 male CAD patients. Coronary plaque properties were evaluated using iMAP®-intravascular ultrasound. Serum anti-LPL Ab levels were not correlated with plaque burden but were significantly negatively and positively correlated with fibrotic and necrotic plaques, respectively. High-risk patients with low anti-apoB-100 Ab levels were divided into groups according to their anti-LPL Ab levels. The group with high anti-LPL Ab levels exhibited more necrotic plaques and fewer fibrotic plaques as well as higher remnant-like lipoprotein particle levels than the group with low anti-LPL Ab levels. Serum anti-LPL Ab levels can serve as a marker of plaque instability in CAD patients and can help identify higher-risk cases when combined with anti-apoB-100 Ab levels.

The Frequency of Celiac Disease and Its Association with Infertility in Women Attending Infertility Clinics: A Case-Control Study in Southern Iran

Background: Celiac disease (CD) is a multifactorial systemic disease that causes enteropathy and can lead to a wide spectrum of disorders related to infertility. Objectives: This case-control study aimed to evaluate the frequency of celiac disease (CD) and its association with infertility in women. Methods: This study was conducted on women referred to Ghadir Mother and Child Hospital in Shiraz. Women with infertility served as the case group, while fertile women comprised the control group. Blood samples were collected from participants, and tissue anti-transglutaminase (Anti-TTG Ab) levels were measured. Patients with elevated Anti-TTG Ab levels were referred for duodenal biopsy. Results: One hundred subjects were enrolled in the case group and 200 in the control group. Eight patients in the case group tested positive for serology, and four of these had duodenal biopsies confirming CD. In the control group, one individual tested positive for serology, but the duodenal biopsy was negative (P for between-group differences: 0.001 for serology and 0.012 for biopsy results). There was a significant association between high levels of Anti-TTG Ab and infertility (odds ratio = 17.30, 95 % CI: 2.13 - 140.39), which remained even after adjusting for age and body mass index (odds ratio = 9.92, 95 % CI: 1.17 - 84.21). Conclusions: The frequency of CD was higher among infertile women compared to fertile women. Increased levels of Anti-TTG Ab were independently associated with infertility.

Lower magnitude and faster waning of antibody responses to SARS-CoV-2 vaccination in anti-TNF-α-treated IBD patients are linked to lack of activation and expansion of cTfh1 cells and impaired B memory cell formation

Patients with inflammatory bowel disease (IBD) and healthy controls received primary SARS-CoV-2-mRNA vaccination and a booster after six months. Anti-TNF-α-treated patients showed significantly lower antibody (Ab) levels and faster waning than α4β7-integrin-antagonist recipients and controls. This prospective cohort study aimed to elucidate the underlying mechanisms on the basis of circulating T-follicular helper cells (cTfh) and B memory cells. We measured SARS-CoV-2- Wuhan and Omicron specific Abs, B- and T-cell subsets at baseline and kinetics of Spike (S)-specific B memory cells along with distributions of activated cTfh subsets before and after primary and booster vaccination. Lower and faster waning of Ab levels in anti-TNF-α treated IBD patients was associated with low numbers of total and naïve B cells vs. expanded plasmablasts prior to vaccination. Along with their low Ab levels against Wuhan and Omicron VOCs, reduced S-specific B memory cells were identified after the 2nd dose which declined to non-detectable after 6 months. In contrast, IBD patients with α4β7-integrin-antagonists and controls mounted and retained high Ab levels after the 2nd dose, which was associated with a pronounced increase in S-specific B memory cells that were maintained or expanded up to 6 months. Booster vaccination led to a strong increase of Abs with neutralizing capacity and S-specific B memory cells in these groups, which was not the case in anti-TNF-α treated IBD patients. Of note, Ab levels and S-specific B memory cells in particular post-booster correlated with the activation of cTfh1 cells after primary vaccination. The reduced magnitude, persistence and neutralization capacity of SARS-CoV-2 specific Abs after vaccination in anti-TNF-α-treated IBD patients were associated with impaired formation and maintenance of S-specific B memory cells, likely due to absent cTfh1 activation leading to extra-follicular immune responses and diminished B memory cell diversification. These observations have implications for patient-tailored vaccination schedules/vaccines in anti-TNF-α-treated patients, irrespective of their underlying disease. The study was funded by third party funding of the Institute of Specific Prophylaxis and Tropical Medicine at the Medical University Vienna. The funders had no role in study design, data collection, data analyses, interpretation, or writing of report.

Abstract 5067: Inhibition of CD47-SIRPα axis to enhance immunity against different molecular subtypes of breast cancer

Abstract Background: The CD47-SIRPα axis is an innate immune checkpoint that primarily regulates myeloid cells. In the context of cancer, tumor cells upregulate CD47 to engage SIRPα inhibitory signaling to block phagocytosis. Thus, inhibition of this axis may enhance myeloid cell phagocytosis and adaptive immune responses against cancer. While our previous studies identified that CD47 mAbs enhance HER2-specific mAb therapy (Tsao et al., 2019), clinical trials have revealed that CD47-targeting mAbs are associated with severe side effects of anemia and thrombocytopenia in patients. We thus hypothesized that targeting SIRPα, which is more restrictively expressed on myeloid cells, may achieve similar but less toxic treatment effects. Methods: To ascertain the impact of SIRPα inhibition against different molecular subtypes of breast cancer, we utilized and validated a novel SIRPα KO mouse, along with SIRPα mAbs in our previously utilized models of Triple-Negative Breast Cancer (TNBC) and HER2+ Breast Cancer (Crosby et al., 2018; Tsao et al., 2019). Using both in vitro and in vivo tumor models, we tested the therapeutic impact of SIRPα inhibition/blockade combined with immunogenic chemotherapy (doxorubicin) in TNBC or combined with HER2-specific mAbs in HER2+ Breast Cancer (HER2+ BC). Results: After validating the loss of SIRPα in KO mice, we found that E0771 TNBC cells had reduced rates of engraftment in SIRPα KO mice compared to controls, but comparable rates of growth once tumors were established. We then treated E0771 engrafted SIRPα KO and wildtype mice with doxorubicin. This treatment elicited tumor rejection in the majority of SIRPα KO mice (70% of injected tumors), compared to only 10% rejection in SIRPα WT mice. To study tumor antigen-specific immunity, we expressed different forms of OVA (secreted, cytoplasmic, membrane) in tumor cells. We found that membrane expressed OVA led to universal tumor rejection (10/10 mice) with high levels of OVA-specific Abs, in contrast to non-membrane expressed forms of OVA. This suggested the importance of tumor-specific Abs in eliciting tumor rejection. To specifically test the antitumor effect of tumor-specific antibodies in the context of SIRPα blockade, we evaluated the impact of a HER2 mAb (Trastuzumab) on HER2+ BC cells. We found enhanced phagocytosis and antitumor effects by SIRPα KO macrophages in comparison to wild-type macrophages, suggesting the potential of targeting SIRPα along with the use of standard-of-care mAb. Conclusion: Our study demonstrates that SIRPα loss can prevent tumor engraftment but may not have a strong effect against established tumors without secondary immune stimulation. However, addition of immunogenic cell death-eliciting chemotherapy or tumor-specific Abs promote tumor phagocytosis and stimulate anti-tumor immunity. These results support further investigation of SIRPα-targeting combination therapies for cancer. Citation Format: Xingru Ma, Zachary Hartman, Li-Chung Tsao, Tao Wang, Cong-Xiao Liu, Xiao Yang, Gangjun Lei, Junping Wei. Inhibition of CD47-SIRPα axis to enhance immunity against different molecular subtypes of breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5067.

Association of High TPO Antibody Titer with Differentiated Thyroid Carcinoma: A Case-Control Study

Background: The most common well-differentiated thyroid carcinomas are papillary thyroid carcinoma (PTC) and follicular thyroid carcinoma (FTC). PTC accounts for 80–90% of all thyroid cancers, and follicular carcinoma accounts for 10–15% of all thyroid cancers. Common established risk factors for thyroid carcinoma are head-neck irradiation, a positive family history, and a few genetic disorders. High anti-thyroglobulin antibodies (TgAb) have already been established as predictive markers for thyroid carcinoma, but anti-thyroid peroxidase antibodies (TPO Ab) have not been established as predictive markers till now. Objective: To find out the association of high TPO Ab titer with differentiated thyroid carcinoma (DTC). Patients and Methods: This case-control study was carried out at the National Institute of Nuclear Medicine & Allied Sciences (NINMAS), Dhaka, from September 2020 to November 2021. A total of 52 cytologically diagnosed DTC patients who underwent thyroidectomy were selected as cases. Another 52 age- and sex-matched healthy individuals who had sonographically normal thyroid glands and a biochemically euthyroid state were selected as controls. The total number of participants was 104. Written informed consent was obtained from each of the participants. A detailed history was taken, and a thorough clinical examination was carried out on each patient. High resolution ultrasonography (HRUS) of the thyroid gland, serum thyroid-stimulating hormone (TSH) level, and fine needle aspiration cytology (FNAC) report of the thyroid nodule of each participant were collected. Serum TPO antibody levels were measured in patients with DTC prior to thyroid surgery, as well as in the control group, in the NINMAS laboratory using the CLIA method. After the collection of all the required data, analysis was done by SPSS 24.0. Results: The mean age of the cases was 35.8 ± 13.3 years and 34.6 ± 13.0 years in the control groups. Female patients were predominant in both the case and control groups (63.5% and 69.2%, respectively). Both groups were statistically almost similar in terms of age and sex. Around 46.2 % (n=24) patients in the case group had high serum TPO Ab levels and 15.4 % (n=8) of participants in the control group had high serum TPO Ab levels. The mean serum TPO Ab level in the case group was 306.77± 452.57 U/mL, and the mean serum TPO Ab level in the control group was 75.85±181.47 U/mL. Serum TPO antibody level was significantly higher in the case group compared to the control group (306.77±452.57 vs 75.85±181.47 U/mL, p = 0.001). Conclusion: There was a significant association of high TPO Ab titer found with DTC Bangladesh J. Nuclear Med. 26(1): 34-38, 2023

Quantification of Severe Acute Respiratory Syndrome Coronavirus 2 Binding Antibody Levels To Assess Infection and Vaccine-Induced Immunity Using WHO Standards.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding antibody (Ab) levels following vaccination or natural infection could be used as a surrogate for immune protection if results of serological assays were standardized to yield quantitative results using an international standard. Using a bead-based serological assay (Luminex xMAP), anti-receptor binding domain (anti-RBD) Ab levels were determined for 1,450 participants enrolled in the Los Angeles Pandemic Surveillance Cohort (LAPSC) study. For 123 participants, SARS-CoV-2 binding antibody unit (BAU) levels were also quantified using WHO standards and then compared to the semiquantitative results. Samples were chosen to represent the range of results and time from vaccination. Antibody levels and decay rates were then compared using unadjusted and adjusted linear regression models. The linear range of the assay used in this study was determined to be 300 to 5,000 mean fluorescence intensity units (MFI). Among the fully vaccinated groups (vaccinated only and vaccinated with past infection), 84.8% had anti-RBD MFI values above the linear range of >5,000 MFI, and 33.8% had values of >15,000 MFI. Among vaccinated participants with past infection (hybrid immunity), 97% had anti-RBD values of >5,000 MFI and 70% (120/171) had anti-RBD values of >15,000 MFI. In the subgroup quantified using the WHO control, BAU levels were significantly higher than the semiquantitative MFI results. In vaccinated participants, Ab decay levels were similar between infected and noninfected groups (P = 0.337). These results demonstrate that accurate quantitation is possible if standardized with an international standard. BAU can then be compared over time or between subjects and would be useful in clinical decision making. IMPORTANCE Accurate quantification of SARS-CoV-2-specific antibodies can be achieved using a universal standard with sample dilution within the linear range. With hybrid immunity being now common, it is critical to use protocols adapted to high Ab levels to standardize serological results. We validated this approach with the Los Angeles Pandemic Surveillance Cohort by comparing the antibody decay rates in vaccinated participants and vaccinated infected participants.

LDH as an adjuvant makes Brucella outer-membrane vesicles and outer-membrane vesicle-associated proteins highly protective in mice.

Existing Brucella vaccines are attenuated and can cause vaccine-associated brucellosis; and these safety concerns have affected their application. Although subunit vaccines have the advantages of safety, efficacy, low cost, and rapid production, they are usually poorly immunogenic and insufficient to trigger persistent immunity. Therefore, we added layered double hydroxide (LDH) as an adjuvant to Brucella subunit vaccine formulations to enhance the immune response to the antigen. LDH and Freund's adjuvant were combined with Brucella outer-membrane vesicles (OMVs) and OMV-associated proteins to form a subunit vaccine, respectively. The immunogenicity of LDH as an adjuvant was assessed in BALB/c mice. We examined levels of immunoglobulin G, G1, and G2a (IgG, IgG1, and IgG2a) antibodies (aBs); percentages of Cluster of Differentiation 4-positive (CD4+) and CD8+ T cells in peripheral-blood lymphocytes; and secretion of cytokines in mouse spleen lymphocytes. Finally, splenic index and splenic bacterial load were assessed via Brucella challenge experiments on mice. The LDH subunit vaccine also produced high levels of specific aBs in mice compared with Freund's adjuvant subunit vaccine and induced mainly T-helper 1 cell (Th1)-type immune responses. In addition, mice in the LDH subunit vaccine group had significantly lower bacterial loads in their spleens than those in the Freund's adjuvant subunit vaccine group, and the LDH-OMV vaccine offered a higher level of protection against Brucella attack. LDH as an adjuvant-paired vaccine provided a high level of protection against Brucella infection.

Longitudinal Randomized Cohort Study of SARS-CoV-2 Antibody Seroprevalence in the St. Petersburg Population.

Introduction. Since the detection of the first COVID-19 patient, 2 years have passed, during which more than 287,862,000 people have fallen ill globally, of which about 1.9% died. The implementation of SARS-CoV-2 control programs required efforts from almost all countries. An important direction in the fight against COVID-19 has been the formation of herd immunity, the main tool for managing the pandemic. Study goal. The aim of the study was to assess the seroprevalence of antibodies (Abs) to SARS-CoV-2 nucleocapsid (Nc) and receptor binding domain (RBD) in the St. Petersburg population during the COVID-19 pandemic. Materials and methods. A longitudinal cohort randomized monitoring study of Ab seroprevalence (SARS-CoV-2 Nc, RBD) was organized and conducted according to a unified methodology developed by Rospotrebnadzor with the participation of the St. Petersburg Pasteur Institute. For this purpose, a cohort was formed of 1000 volunteers who participated in all five stages of seromonitoring. The cohort was divided into seven age groups: 1–17; 18–29; 30–39; 40–49; 50–59; 60–69; 70; and older (70+) years. Seropositivity levels (Nc, RBD) were assessed by quantitative and qualitative enzyme immunoassays. During the second year of monitoring, some volunteers were vaccinated with the GamCOVIDVac (84%) or EpiVacCorona (11.6%) vaccines approved in Russia. Statistical processing was carried out using Excel 2010. Confidence intervals for shares and percentages (95% CI) were calculated using the method of A. Wald and J. Wolfowitz with adjustment (A. Agresti, B.A. Coull). The statistical significance of differences was calculated by z-test, using the appropriate online calculator (p < 0.05) unless indicated. Results. There was a trend toward an increase in Nc seropositivity in stages 1–3 of seromonitoring, with a decrease in stages 4–5 among children and adults. The share of RBD seropositive steadily increased during all five stages of seromonitoring. The most frequent finding was low anti-RBD Abs levels (22.6–220 BAU/mL). High Ab levels were recorded statistically significantly less frequently. Asymptomatic forms were observed in 84–88% of SARS-CoV-2 seropositive volunteers. By the fifth stage of monitoring, this indicator significantly decreased to 69.8% (95% CI: 66.1–73.4). The monitoring revealed a statistically significant increase in anti-RBD Abs alongside a statistically significant decrease in the proportion of Nc seropositives. This dynamic was especially characteristic of persons vaccinated with GamCOVIDVac. Conclusion. Prior to the use of specific vaccines, a seroprevalence of anti-Nc Abs was noted. After the introduction of the GamCOVIDVac vaccine in adults, a decrease in the level of anti-Nc Abs was noted due to an increase in the proportion of RBD seropositive persons.

27. Immunologic Hyporesponsiveness with Subsequent Dosing of Meningococcal Vaccines: Re-Evaluating the Current Paradigm

BackgroundImmunologic hyporesponsiveness (HyR) is considered as an inability to mount immune responses to vaccination of at least the same degree as earlier doses. For meningococcal vaccines, HyR has classically been associated with unconjugated but not conjugated polysaccharide (PS) vaccine dosing, but the clinical relevance is unclear. MethodsTo characterize meningococcal vaccine HyR, a PubMed search was conducted without date limits as follows: (hyporespons*) AND (meningococcal) AND (vaccine OR mechanism OR MOA OR causes). Papers from the authors’ files, including HyR insights with other vaccines, were included. ResultsClassic HyR with repeat unconjugated PS vaccine (MPV) dosing is thought to be associated with memory B-cell (BC) depletion, causing reduced responses on redosing with the same PS. This lack of immunologic memory and interference is seen years after MPV dosing across age groups. As data is added, other examples seem to fit the HyR definition but differ from the classical mechanism and its implications. First, passively transferred maternal antibodies (Abs) may interfere with neonatal adaptive immune response and ultimately those of childhood vaccination by binding to vaccine antigen (Ag) and inhibiting Ab production. Second, multiple dose schedules of meningococcal conjugate vaccines can show reduced responses to later doses in the series but memory is still established and amnestic booster response later achieved. Finally, carrier-induced epitopic suppression, occurring when PS Ag epitopes presented on a protein carrier are inhibited by prior/concurrent dosing with the same carrier, has also been reported. These 3 examples of alternative HyR mechanisms are not associated with memory BC depletion but are likely due to high circulatory Ab levels reducing responses, which is transient, reduces with Ab waning, immunologic memory remains intact, and is not clinically significant.ConclusionThis literature review identified HyR mechanisms other than the classic mechanism associated with memory BC depletion that may account for decreased immune response to subsequent vaccination. Understanding the type of HyR observed with meningococcal vaccines is crucial, as these mechanisms vary in terms of potential clinical significance and the duration of their impact.Disclosures Jamie Findlow, PhD, Pfizer (Employee, Shareholder) Paul Balmer, PhD, Pfizer (Employee, Shareholder)

Post-Covid-19 Hemostatic Characteristics of Convalescent Patients: The Bergamo Experience

Introduction: Severe COVID-19 patients present with a hypercoagulable state, complement activation and endothelial perturbation, which result from an excessive inflammatory response. Thromboinflammation is one important mechanism underlying the COVID-19-associated coagulopathy and the increased risk of thrombosis. Bergamo city is one of the first and most affected area by SARS-CoV-2 infection in the world. For this reason, since the beginning we were actively involved in recruiting convalescent COVID-19 patients, in a program of selection of candidates for convalescent plasma donation. In a large cohort of convalescent COVID-19 patients, we aimed to characterize markers of coagulation activation and endothelial perturbation, in order to explore whether the COVID-19-related hemostasis activation might persist afterwards and evaluate its possible association with the degree of severity of the previous infection, and/or with demographic characteristics, or anti-SARS-CoV-2 antibody levels.Methods: In 392 convalescent COVID-19 patients (216M/176F, median age: 46 years) plasma levels of fibrinogen, protein C, protein S, factor V, factor VIII, factor XIII, D-dimer, von Willebrand factor (vWF), prothrombin fragment F1+2 were measured at the recruitment, i.e. 1-5 months from recovery. Samples were tested for the anti-SARS-CoV-2 antibodies, including anti-S IgG (Anti-S Ab) and anti-N IgG (Anti-N Ab) antibodies at enrollment and at each scheduled subsequent visits.Results: Levels of fibrinogen, D-dimer, von Willebrand factor, protein S and protein C were significantly higher (p<0.05) in patients who were hospitalized for severe COVID-19 as compared to patients who were treated at home. There was no correlation between levels of coagulation biomarkers and days from end of symptoms. Male gender, age > 40 years, and severe form of COVID-19 were identified as independent predictors of high levels of both anti-S and anti-N Ab (p<0.001). Among hemostatic biomarkers, fibrinogen (p<0.01) and vWF (p<0.05) independently predict high levels of anti-S Ab. In particular, vWF levels positively correlated with anti-S Ab levels (vWF-antigen r=0.188; vWF-activity r=0.241 and vWF-RiCof r=0.223, p<0.01). Evaluation of anti-SARS-CoV2 antibody levels at different time points during follow up revealed that 30% of patients displayed high levels of anti-S Ab until more than 8 months from the end of symptoms.Conclusions: Convalescent patients, with a history of severe COVID-19 had a persistent endothelium activation, despite of disease clinical remission even after 9 months from end of symptoms. Furthermore, fibrinogen and vWF levels predicted high levels of Anti-S Ab. Among demographic characteristics, gender, age and severe disease can be predictors of increased antibody response. These findings suggest that inflammation, coagulation and endothelial dysfunction may persist after recovery and may explain the findings of persistent clinical symptoms reported in these patients after healing from COVID-19. DisclosuresFalanga: Bayer: Honoraria; Leo Pharma: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria.

Herd immunity to SARS-CoV-2 among the population of the Republic of Belarus amid the COVID-19 pandemic

Objective was to investigate the SARS-CoV-2 collective immunity status of the population of Belarus within the context of the COVID-19 pandemic. Materials and methods. The work was carried out according to the methodology for assessing SARS-CoV-2 population immunity, developed by Rospotrebnadzor Russia and the Ministry of Health of Belarus with the participation of the St. Petersburg Pasteur Institute, taking into account WHO recommendations. The Bioethics Committee of Belarus and the local ethics committee of the St. Petersburg Pasteur Institute approved the study. Selection of participants was carried out using a questionnaire method and online technology (internet, cloud server). Volunteers were randomized into seven age groups (years of age): 1–17; 18–29; 30–39; 40–49; 50–59; 60–69; and 70+. Regional randomization ensured proportional representation of volunteers from each region, and no more than 30 people were included from one enterprise. In accordance with manufacturer instructions, blood plasma samples were analyzed for: IgG antibodies (Abs) to the SARS-CoV-2 nucleocapsid (Nc) using a quantitative ELISA test system; and IgG Abs to the receptor binding domain (RBD) of the SARS-CoV-2 S (spike) surface glycoprotein using a qualitative ELISA test system. Statistical processing was carried out using Excel 2010 and other software. Statistical differences were designated as significant when p 0.05, unless otherwise indicated. Results. The level of seroprevalence, in terms of Abs to Nc among the Belarusian population, was 38.4% (95% CI 37.6–45.4). The highest Ab levels were found among individuals in older age groups (50-70+ years old). The lowest were found in children 1–17 years old and in young people 18–39 years old The distribution of seroprevalence across Belarusian regions was relatively homogeneous, with the exception of the Minsk Region, where a statistically significant decrease in the indicator was noted. In terms of profession, the largest share of seropositive individuals was found among transportation workers; the smallest was found in business. The moderate COVID-19 incidence has not led to a dramatic increase in the number of contacts. The base reproduction number (R0) was 1.3. In the Republic of Belarus, there was a moderate level of asymptomatic COVID-19 among seropositive individuals (45.3% [95% CI 44.0–46.7]). This form of infection was observed most often among children aged 1–17 years old (65.0% [95% CI 61.3–68.6]). In parallel with seroprevalence assessment, SARS-CoV-2 vaccination was carried out. We used two vaccines: Gam-COVID-Vac (also known as Sputnik V, developed by Gamaleya National Center for Epidemiology and Microbiology, Russia); and BBIBP-CorV (Sinopharm, PRC). Vaccination against SARS-CoV-2 was accompanied by an increase in the level of anti-RBD Abs (95% [95% CI 94.7–96.7]). Taking into account the vaccination of a subset of the population with BBIBP-CorV, the overall herd immunity, inferred from the analyzed indicators (presence of anti-Nc or anti-RBD Abs), was 47.1% (95% CI 46.3–48.0). Conclusion. COVID-19 in Belarus was characterized by a moderately pronounced course of the epidemic process. The threshold level of herd immunity to SARS-CoV-2 has not yet been reached, as a result of which the conditions for progression of the epidemic remain.

Analytical and clinical evaluation of four commercial SARS-CoV-2 serological immunoassays in hospitalized patients and ambulatory individuals

BackgroundThis study aimed to compare four anti-SARS-CoV-2 immunoassays in populations presenting different clinical severity levels. MethodsThree populations were included: “severe-to-critical” ICU-hospitalized patients (n = 18), “mild-to-moderate” hospitalized patients (n = 16) and non-hospitalized symptomatic patients (n = 24). Four commercial immunoassays were analyzed and validated: anti-IgG ARCHITECT® (Abbott), anti-Total antibodies (Ab) VITROS® (Ortho Clinical Diagnostics), anti-IgG NovaLisa® (NovaTec Immundiagnostica) and Healgen® IgM and IgG (Zhejiang Orient Gene Biotech). Sensitivities were evaluated according to days post-symptoms onset (pso). Specificities were evaluated on SARS-CoV-2-negative control sera collected before January 2020. ResultsA majority of severe-to-critically ill patients showed detectable Ab already at day 14 and sensitivities reached 100 % after 22 days pso. For patients with “mild-to-moderate” illness, sensitivities increased by at least 5-fold from day 0 to day 14 pso. Non-hospitalized symptomatic individuals already seroconverted at day 14 days pso with 100 % sensitivities for Total Ab VITROS®. Specificities were evaluated at 97 % for ARCHITECT® and NovaLisa®, 98 % for VITROS® and at 94 % for Healgen® combined IgM and IgG. Five “severe-to-critically” ill patients presented high positive Ab levels for at least 16 weeks pso. ConclusionThe Ab levels and the evaluated sensitivities, representing the true positive rate, increased overtime and were related to the COVID-19 severity. Automated Total Ab immunoassay showed better sensitivities and specificity for immunological surveillance and vaccine evaluation.

Enolase-specific cross antibodies induce neutrophilic inflammation in the intestine.

The pathogenesis of ulcerative colitis (UC) is to be further investigated. House dust mites (HDM) are highly associated with the pathogenesis of immune inflammation in the body. This study aims to investigate the role of enolase (one of the HDM-derived proteins)-specific cross Abs in the induction of UC-like inflammation. The enolase specific IgG (EsIgG) was identified in UC patients by mass spectrometry. Mice were treated with EsIgG to induce inflammation in the colon mucosa. EsIgG was detected in the serum and the colon tissues of UC patients, which was positively correlated with the polymorphonuclear neutrophil (PMN) counts in the blood and colon tissues of UC patients. EsIgG formed immune complexes with the constitutive enolase in the UC colon epithelium that activated complement, induced epithelial cell apoptosis, compromised epithelial barrier functions, and resulted in UC-like inflammation in the mouse colon. In summary, UC patients have high serum levels of Abs against HDM-derived enolase and intestinal epithelial cell-derived enolase. These Abs attack the colonic epithelium to induce UC-like inflammation.

P0382“FALSE” POSITIVITY OF SERUM ANTI-PHOSPHOLIPASE A2 RECEPTOR ANTIBODY (ANTI-PLA2R AB) IN PATIENTS WITH NON MEMBRANOUS GLOMERULAR DISEASE

Abstract Background and Aims Phospholipase A2 receptor (PLA2R) is the major target antigen in primary membranous nephropathy (PMN) and the detection of circulating autoantibodies to PLA2R (Anti-PLA2R Ab) is a major advance in understanding this disease. In this study, we aimed to determine if Anti-PLA2R Ab serves as a specific marker for PMN in our multi-ethnic Southeast Asian cohort. Method This was a prospective cohort study of all adults with glomerular disease undergoing biopsy between 1st January 2015 and 30th June 2018. Anti-PLA2R antibody levels were quantitatively determined by IgG ELISA (Euroimmun). Results are expressed as RU/ml. We considered a value of &amp;gt;20RU/ml to represent a positive result, value of &amp;lt;14RU/ml to be negative and values between 14-20 RU/ml to be indeterminate. Results Out of the cohort of 192 patients, there were 27 patients with Membranous Nephropathy (MN), with 12 PMN and 15 with secondary MN (SMN). Secondary causes were Hepatitis (1), Malignancy (1), Lupus (13). In our analyses, 10 (83.3%) PMN pts were positive for Anti-PLA2R Ab, with 7(58.3%) strongly positive with titres &amp;gt;200RU/ml and 2(16.7%) negative. For SMN, the majority, 12 out of 14 patients (85.7%) tested negative and the remaining 2 (7.7%) yielded values of indeterminate levels. Of note, among the 165 patients with non membranous glomerular disease, there were 15 patients (9.1%) with indeterminate Anti-PLA2R Ab levels and 31 patients (18.9%) tested positive for Anti-PLA2R antibody, with levels &amp;gt;20 RU/ml. Of these 31 patients, the majority of the patients had low levels of Anti-PLA2R Ab of &amp;lt;50 RU/ml. However there were 5 patients who had considerably high levels of Anti-PLA2R Ab of &amp;gt;50 RU/ml and their characteristics are tabled below (Table 1) Conclusion Consistent with other published studies, positive Anti-PLA2R antibody levels were highly specific for PMN in our multi-ethnic Southeast Asian population and our centre has since been consistently using this test for differentiating between PMN and SMN. What is most striking in our study is that we have found high-titre Anti-PLA2R antibody positivity in non-MN patients, which has not been frequently reported in literature. The unifying feature amongst these patients appear to be the presence of arterial and arteriolar sclerosis which is usually non-specific. Though we would not normally do the Anti-PLA2R antibody test in a patient with low suspicion of MN, our study findings warrants further investigation and may indicate the limited usefulness of Anti-PLA2R antibody levels as a screening tool for glomerulonephritis.

1212P - Comprehensive results of a phase Ib study with a HER2/neu B-cell peptide vaccine administered with cisplatin and 5-fluorouracil or capecitabine chemotherapy show safety, immunogenicity and clinical response in patients with HER2/Neu overexpressing advanced gastric cancer

BackgroundHER2/neu is overexpressed in 15-25% of gastric cancer patients and associated with poor prognosis. Treatment with recombinant monoclonal Abs against HER2/neu is effective yet alternatives are needed due to cost and global availability issues. Thus a peptide vaccine (IMU-131) was developed, consisting of 3 fused B-cell epitopes (p467) from the HER2/neu extracellular domain coupled to CRM197 and administered with the adjuvant Montanide. The present study evaluated the optimal/safe vaccine dose leading to immunogenicity and clinical responses. MethodsIn an open-label multicenter Phase Ib trial (SE-Asia & Eastern Europe), 14 patients with HER2/neu overexpressing (++/+++) gastric adenocarcinoma were recruited to receive 3 injections of IMU-131 (days 0, 14, 35) in combination with chemotherapy (CT). Dose escalation (10, 30 and 50 µg) was performed in 3 cohorts (C) to evaluate safety, immunogenicity and clinical responses. ResultsNo SAEs related to IMU-131 administration were reported. Eleven patients were evaluable for vaccine-specific immune responses and RECIST assessment. Higher HER2-specific IgG levels were observed in C2 (30 µg) vs. C1 (10 µg). 3/5 patients in C2 showed moderate or little increase in HER2-specific Abs, while all C3 patients (50µg) responded with moderate to high Ab levels. According to RECIST, 1 patient showed complete response, 5 partial response and 4 stable disease. Strong correlation between high Ab levels and clinical responses was observed in C3, while this was only moderate in C2. Capacity to inhibit HER2 phosphorylation was tested in patient sera and could be detected in association with strong tumor reduction. Furthermore, patients with good clinical responses, but low Ab titers featured cellular responses with high IFNγ and TNFα/IL-10 ratios. ConclusionsThe vaccine was well tolerated and safe with Ab responses at the highest dose showing strong correlation with clinical responses. Currently, the 50 µg dose is being evaluated in a Phase II trial with two arms (IMU 131 + CT and CT only). Clinical trial identificationNCT02795988. Legal entity responsible for the studyImugene Ltd. FundingImugene Ltd. DisclosureU. Wiedermann: Research grant / Funding (institution), Officer / Board of Directors, CSO until September 2018: Imugene; Research grant / Funding (institution): GSK; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Themis. I. Bulat: Advisory / Consultancy, Research grant / Funding (self), Full / Part-time employment: Arensia Exploratory Medicine . T. Yau: Honoraria (self), Advisory / Consultancy: Bristol-Myers Squibb. M. Maglakelidzde: Advisory / Consultancy, Research grant / Funding (self), Full / Part-time employment: ARENSIA Exploratory Medicine . T. Ungtrakul: Travel / Accommodation / Expenses: AstraZeneca. C.C. Zielinski: Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): BMS; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): MSD; Honoraria (self), Advisory / Consultancy, Officer / Board of Directors, Scientific Advisory Board Member until June 2018: Imugene; Honoraria (self), Advisory / Consultancy: Ariad; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Honoraria (self), Advisory / Consultancy: Merrimack; Honoraria (self), Advisory / Consultancy: Merck; Honoraria (self), Advisory / Consultancy: KGaA; Honoraria (self), Advisory / Consultancy: Fibrogen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Honoraria (self), Advisory / Consultancy: Tesaro; Honoraria (self), Advisory / Consultancy: Gilead; Honoraria (self), Advisory / Consultancy: Servier; Honoraria (self), Advisory / Consultancy: Shire; Honoraria (self), Advisory / Consultancy: Eli Lilly; Honoraria (self), Advisory / Consultancy: Athenex. L. Chong: Honoraria (self), Advisory / Consultancy, Leadership role, Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Full / Part-time employment: Imugene Ltd. N. Ede: Leadership role, Research grant / Funding (institution), Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Licensing / Royalties, Full / Part-time employment: Imugene Ltd. A. Good: Advisory / Consultancy, Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: Imugene Ltd; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Kazia Therapeutics; Shareholder / Stockholder / Stock options: Living Cell Technologies. All other authors have declared no conflicts of interest.

Immunization against environmental chemical carcinogens: Pro and contra

The problems of immunoprotection from the environmental chemical carcinogens are discussed. The main experimental argument pro active immunization against carcinogens is a possibility of specific mucosal antibodies (Abs) to inhibit the penetration of carcinogens from environment and to stimulate its excretion with the following decreasing of carcinogen-DNA adducts levels. Hypothesis of cancer immunostimulation after active immunization against carcinogens is based on a high cancer risk in persons with high levels of serum Abs specific to environmental carcinogens coupled with high levels of Abs to endogenous steroids stimulating the proliferation of target cells, for example, Abs to benzo[a]pyrene together with Abs to estradiol. The active immunization could increase the cancer risk much more in those persons. The passive immunization could be an alternative safe approach to avoid this problem.

Chimeric Hemagglutinin-Based Influenza Virus Vaccines Induce Protective Stalk-Specific Humoral Immunity and Cellular Responses in Mice.

The high variation of the influenza virus hemagglutinin (HA), particularly of its immunodominant head epitopes, makes it necessary to reformulate seasonal influenza virus vaccines every year. Novel influenza virus vaccines that redirect the immune response toward conserved epitopes of the HA stalk domain should afford broad and durable protection. Sequential immunization with chimeric HAs (cHAs) that express the same conserved HA stalk and distinct exotic HA heads has been shown to elicit high levels of broadly cross-reactive Abs. In the current mouse immunization studies, we tested this strategy using inactivated split virion cHA influenza virus vaccines (IIV) without adjuvant or adjuvanted with AS01 or AS03 to measure the impact of adjuvant on the Ab response. The vaccines elicited high levels of cross-reactive Abs that showed activity in an Ab-dependent, cell-mediated cytotoxicity reporter assay and were protective in a mouse viral challenge model after serum transfer. In addition, T cell responses to adjuvanted IIV were compared with responses to a cHA-expressing live attenuated influenza virus vaccine (LAIV). A strong but transient induction of Ag-specific T cells was observed in the spleens of mice vaccinated with LAIV. Interestingly, IIV also induced T cells, which were successfully recalled upon viral challenge. Groups that received AS01-adjuvanted IIV or LAIV 4 wk before the challenge showed the lowest level of viral replication (i.e., the highest level of protection). These studies provide evidence that broadly cross-reactive Abs elicited by cHA vaccination demonstrate Fc-mediated activity. In addition, cHA vaccination induced Ag-specific cellular responses that can contribute to protection upon infection.

ABO Tolerance Following Treatment of Infant Mice with A-Expressing MHC-Identical Erythrocytes

Purpose ABO-incompatible heart transplantation (ABOi HTx) is safe in young children and increases donor access. Post-ABOi HTx, B cell tolerance develops to donor ABO blood group antigen(s) by mechanisms not fully defined. To study ABO tolerance, we developed A-transgenic mice (A-Tg) that express A-antigen on vascular endothelium, erythrocytes (RBCs) and lymphocytes; we demonstrated A-antigen-specific tolerance induced by HTx into 4 wk-old, MHC-identical, wild-type (WT) mice. Intentional induction of tolerance may allow subsequent ABOi HTx. Recently, we showed ABO tolerance could be intentionally induced in WT mice after ip injection of infant mice with intact A-Tg blood cells (BCs). Herein, we sought to determine specific A-Tg cell type(s) capable of inducing tolerance. Methods WT BALB/c (BALB) mice at 7 days of age were left untreated or injected ip (weekly × 3) with either unfractionated BCs, erythrocytes (RBCs), peripheral blood mononuclear cells (PBMCs), or splenocytes from A-Tg BALB mice (see Table). Two weeks later, all mice were injected ip (weekly × 5) with human A-RBC (A-antigen challenge) in an attempt to elicit anti-A antibody (Ab) production. Serum anti-A and third-party (non-A anti-human) Ab were assessed by hemagglutination or ABH glycan microarray. Results In response to challenge with A-antigen, high levels of anti-A Ab were produced both in untreated mice and in mice previously treated with A-Tg PBMCs or splenocytes (Table). In contrast, anti-A Ab remained undetectable/very low in mice treated as infants with A-Tg BCs or RBCs. Third-party Ab responses were high for all groups. Conclusion A-Tg RBCs induced robust A-antigen-specific tolerance in infant mice, whereas A-Tg lymphocytes (PBMCs, splenocytes) did not. Future studies will explore mechanisms of A-Tg RBC tolerance induction with the goal to design synthetic ABH-multivalent polyethylene glycol (PEG) glycoconjugates for intentional ABO tolerance to allow subsequent ABOi HTx safely.

The Magnitude and IgG Subclass of Antibodies Elicited by Targeted DNA Vaccines Are Influenced by Specificity for APC Surface Molecules.

Upon APC-targeted DNA vaccination, transfected cells secrete fusion proteins with targeting units specific for surface molecules on APC. In this study, we have tested several different targeting units for their ability to influence the magnitude and subclass of Ab responses to hemagglutinin from influenza A virus. The experiments employed bivalent homodimeric Ig-based molecules (vaccibodies). The overall efficiency in BALB/c mice depended on the targeting units in the following order: αMHC class II > αCD11c > αCD40 > Xcl-1 = MIP-1α > FliC > GM-CSF > Flt-3L > αDEC205. GM-CSF induced mainly IgG1, whereas Xcl1, MIP-1α, αCD40, and αDEC205 induced predominantly IgG2a. A more balanced mixture of IgG1 and IgG2a was observed with αCD11c, αMHC class II, Flt-3L, and FliC. Similar results of IgG subclass-skewing were obtained in Th1-prone C57BL/6 mice with a more limited panel of vaccines. IgG1 responses in BALB/c occurred early after immunization but declined relatively rapidly over time. IgG2a responses appeared later but lasted longer (>252 d) than IgG1 responses. The most efficient targeting units elicited short- and long-term protection against PR8 influenza (H1N1) virus in BALB/c mice. The results suggest that targeting of Xcr1+ conventional type 1 dendritic cells preferentially induces IgG2a responses, whereas simultaneous targeting of several dendritic cell subtypes also induces IgG1 responses. The induction of distinct subclass profiles by different surface molecules supports the APC-B cell synapse hypothesis. The results may contribute to generation of more potent DNA vaccines that elicit high levels of Abs with desired biologic effector functions.

Enhancing vaccine antibody responses by targeting Clec9A on dendritic cells

Targeting model antigens (Ags) to Clec9A on DC has been shown to induce, not only cytotoxic T cells, but also high levels of Ab. In fact, Ab responses against immunogenic Ag were effectively generated even in the absence of DC-activating adjuvants. Here we tested if targeting weakly immunogenic putative subunit vaccine Ags to Clec9A could enhance Ab responses to a level likely to be protective. The proposed “universal” influenza Ag, M2e and the enterovirus 71 Ag, SP70 were linked to anti-Clec9A Abs and injected into mice. Targeting these Ags to Clec9A greatly increased Ab titres. For optimal responses, a DC-activating adjuvant was required. For optimal responses, a boost injection was also needed, but the high Ab titres against the targeting construct blocked Clec9A-targeted boosting. Heterologous prime-boost strategies avoiding cross-reactivity between the priming and boosting targeting constructs overcame this limitation. In addition, targeting small amounts of Ag to Clec9A served as an efficient priming for a conventional boost with higher levels of untargeted Ag. Using this Clec9A-targeted priming, conventional boosting strategy, M2e immunisation protected mice from infection with lethal doses of influenza H1N1 virus.