IMMUNOGRAPH-BASED ANALYSIS OF THE INFLUENZA A (H1N1)PDM09 VACCINE STRAIN IMMUNOGENICITY IN THE PANDEMIC AND POST-PANDEMIC PERIOD (2009-2014)

Currently, the assessment of the immunogenic properties of influenza viruses as a part of influenza vaccines, is carried out by using seroprotection, seroconversion as well as the rate of increases in post-vaccination antibodies. At the same time, significant differences in the immunogenicity of vaccines related to dynamic formation of high antibody titers responsible for long-term protection of the vaccinated, are neglected.Influenza viruses such as A (H1N1) pdm09 that caused 2009-2010 pandemic continue to circulate in the population, therefore, the assessment of the immunogenic activity of vaccine viruses prepared during the pandemic period is interesting in for the methodology to prepare pandemic vaccines to be used in various groups (adults, children, elderly people).Analyzing immunogenicity of influenza vaccines used during the 2009-10 swine influenza pandemic and the post-pandemic period up to the year 2014 was carried out by applying the graphical method for assessing immunogenicity (immunographs) measured as follows: for each group of vaccinated subjects (depending on the vaccine used), an increased rate in antibody level was calculated and the graphs of immunogenicity were plotted. An increased rate of serum antibodies magnitude from vaccinated subjects and the number of sera (in%) with a given fold increase rate in antibody level from 1 to the maximum magnitude were plotted on the x- and y-axis, respectively. The proposed method for assessing immunogenicity allows to plot immunogenicity graphs regardless of the serum antibodies level found in volunteers. The assessment described above revealed a several features for developing immune response to the pandemic virus A (H1N1)pdm09 such as the lack of immune response in a substantial number of adult volunteers (25-27%%) and young children (60-70%%) after monovaccine administration. The reason for such immune response can be both an insufficient dose of vaccine-containing viral antigen and suppressed immune response caused by the influenza A(H1N1)pdm09.A study on the immunogenic properties for seasonal influenza vaccines containing the influenza A (H1N1) pdm09 virus antigen in the years 2010 - 2014 revealed a variety in emerging humoral immunity ranging from a short-term, low-frequency increase in antibodies from vaccinated children to the formation of high antibody titers in elderly.Practically, immunographic analysis of influenza vaccines particularly those derived from the influenza A (H1N1)pdm09 virus, may result in proposing recommendations to increase an antigenic load at the beginning of a pandemic cycle and/or block the suppressive properties of vaccine-contained viruses in pediatric vaccines, because escalating virus dose in the vaccine may not always be achievable in this case.

Antibody in Lymphocyte Supernatant (ALS) responses after oral vaccination with live Shigella sonnei vaccine candidates WRSs2 and WRSs3 and correlation with serum antibodies, ASCs, fecal IgA and shedding

The levels of antigen-specific Antibodies in Lymphocyte Supernatant (ALS) using an ELISA are being used to evaluate mucosal immune responses as an alternate to measuring the number of Antibody Secreting Cells (ASCs) using an ELISpot assay. A recently completed trial of two novel S. sonnei live oral vaccine candidates WRSs2 and WRSs3 established that both candidates were safe, well tolerated and immunogenic in a vaccine dose-dependent manner. Previously, mucosal immune responses were measured by assaying IgA- and IgG-ASC in peripheral blood mononuclear cells (PBMCs). In this report, the magnitude of the S. sonnei antigen-specific IgA- and IgG-ALS responses was measured and correlated with previously described ASCs, serum antibodies, fecal IgA and vaccine shedding. Overall, the magnitude of S. sonnei anti-Invaplex50 ALS was higher than that of LPS or IpaB, and both vaccines demonstrated a more robust IgA-ALS response than IgG; however, compared to WRSs3, the magnitude and percentage of responders were higher among WRSs2 recipients for IgA- or IgG-ALS. All WRSs2 vaccinees at the two highest doses responded for LPS and Invaplex50-specific IgA-ALS and 63–100% for WRSs3 vaccinees responded. Regardless of the vaccine candidate, vaccine dose or detecting antigen, the kinetics of ALS responses were similar peaking on days 7 to 9 and returning to baseline by day 14. The ALS responses were vaccine-specific since no responses were detected among placebo recipients at any time. A strong correlation and agreement between responders/non-responders were noted between ALS and other mucosal (ASC and fecal IgA) and systemic (serum antibody) immune responses. These data indicate that the ALS assay can be a useful tool to evaluate mucosal responses to oral vaccination, an observation noted with trials of other bacterial diarrheal pathogens. Furthermore, this data will guide the list of immunological assays to be conducted for efficacy trials in different populations. It is hoped that an antigen-specific-ALS titer may be a key mucosal correlate of protection, a feature not currently available for any Shigella vaccines candidates. https://clinicaltrials.gov/show/NCT01336699.

Development and Characterization of a Multiplex Assay to Quantify Complement-Fixing Antibodies against Dengue Virus

Antibodies capable of activating the complement system (CS) when bound with antigen are referred to as “complement-fixing antibodies” and are involved in protection against Flaviviruses. A complement-fixing antibody test has been used in the past to measure the ability of dengue virus (DENV)-specific serum antibodies to activate the CS. As originally developed, the test is time-consuming, cumbersome, and has limited sensitivity for DENV diagnosis. Here, we developed and characterized a novel multiplex anti-DENV complement-fixing assay based on the Luminex platform to quantitate serum antibodies against all four serotypes (DENV1-4) that activate the CS based on their ability to fix the complement component 1q (C1q). The assay demonstrated good reproducibility and showed equivalent performance to a DENV microneutralization assay that has been used to determine DENV serostatus. In non-human primates, antibodies produced in response to primary DENV1-4 infection induced C1q fixation on homologous and heterologous serotypes. Inter-serotype cross-reactivity was associated with homology of the envelope protein. Interestingly, the antibodies produced following vaccination against Zika virus fixed C1q on DENV. The anti-DENV complement fixing antibody assay represents an alternative approach to determine the quality of functional antibodies produced following DENV natural infection or vaccination and a biomarker for dengue serostatus, while providing insights about immunological cross-reactivity among different Flaviviruses.

Screening for antibodies against the sheep scab mite (Psoroptes ovis) Pso o 2 antigen in experimentally infested Swifter sheep may fail to identify affected animals

Sheep scab, caused by Psoroptes ovis mites, represents a significant threat to sheep health and welfare. Infestations are diagnosed by parasite identification in skin scrapings, and more recently with a commercial ELISA against serum antibodies to the Pso o 2 mite allergen. However, little is known about the performance of the ELISA in non-UK sheep populations. In this study, six Swifter sheep were experimentally infested with P. ovis. Lesion sizes were monitored and serum IgG against Pso o 2 and the novel Pso-EIP-1 antigens were measured by ELISA. Although all sheep showed signs of infestation, serum from two animals failed to react with Pso o 2. However, they did react to Pso-EIP-1. This indicates that cases of sheep scab in (Swifter) sheep may remain undetected using the Pso o 2 ELISA, which may have implications for routine screening of non- UK sheep breeds.

Highly Specific Memory B Cells Generation after the 2nd Dose of BNT162b2 Vaccine Compensate for the Decline of Serum Antibodies and Absence of Mucosal IgA

Specific memory B cells and antibodies are a reliable read-out of vaccine efficacy. We analysed these biomarkers after one and two doses of BNT162b2 vaccine. The second dose significantly increases the level of highly specific memory B cells and antibodies. Two months after the second dose, specific antibody levels decline, but highly specific memory B cells continue to increase, thus predicting a sustained protection from COVID-19. We show that although mucosal IgA is not induced by the vaccination, memory B cells migrate in response to inflammation and secrete IgA at mucosal sites. We show that the first vaccine dose may lead to an insufficient number of highly specific memory B cells and low concentration of serum antibodies, thus leaving vaccinees without the immune robustness needed to ensure viral elimination and herd immunity. We also clarify that the reduction of serum antibodies does not diminish the force and duration of the immune protection induced by vaccination. The vaccine does not induce sterilizing immunity. Infection after vaccination may be caused by the lack of local preventive immunity because of the absence of mucosal IgA.

Determination of Anti-Phospholipase A2 and Anti-Thrombospondin Type 1 Domain-Containing Protein 7A in Latin Patients With Membranous Nephropathy

BACKGROUND Membranous nephropathy (MN) is caused by antibodies against podocyte antigens, of which the type M receptor of phospholipase A2 (PLA2R) and thrombospondin type-1 domain containing 7 A (THSD7A) are the mostly studied. The objectives of this study were to determine anti-PLA2R and anti-THSD7A serum antibodies and anti-PLA2R renal tissue prevalence in a Latin population with primary MN and to evaluate their role as biomarkers for disease activity. Additionally, the performance of the two available serum diagnostic methods - ELISA and indirect immunofluorescence - was evaluated for the diagnosis of MN. METHODS Fifty-nine patients, 29 MN, 18 lupus membranous nephropathy (LMN) and 12 focal and segmental glomerulosclerosis (FSGS) were evaluated for serum antibodies. Renal biopsies were also evaluated for the presence of anti-PLA2R. Twenty-one patients with MN were followed for 1 year. RESULTS Patients with LMN and FSGS were negative for both antibodies. All 29 MN were negative for anti-THSD7A; 16 MN were positive by ELISA and/or IFI, and 3 MN patients were positive only by IFI. Thus, ELISA test showed a 45% sensitivity and 97% specificity while the IFI showed a 55% and 100%, respectively. In patients with less than a year between biopsy and antibody collection, sensitivity increased to 79% and maintained specificity. Positive correlations were observed between the anti-PLA2R ELISA titer and proteinuria. Among the 28 MN renal biopsies, 20 presented anti-PLA2R positive staining, a 72% sensitivity. CONCLUSIONS The determination of anti-PLA2R antibodies in the Latin population showed similar rates as reported in other populations, and the IFI test presented greater sensitivity. A lower rate of remission was found among those with anti-PLA2R positive tests.