Characterisation of T Follicular Helper Cell (TFH) in Nasopharynx-Associated Lymphoid tissue and Its effect on regulation of immune response to influenza virus

TFH cells have been identified as a new T helper subset specialized to regulate the development of effector and memory B cells and long-lived plasma cells. The interaction between TFH and B cells leads to the activation of B cells and germinal centers (GC) formation. Considering the importance of TFH for B cell antibody response, novel vaccine adjuvants and intranasal vaccines to boost TFH number or function may be an attractive vaccination strategy to enhance vaccine efficacy in humans. Adenotonsillar tissues are major parts of nasopharyngeal associated lymphoid tissues (NALT) and they are important in response to upper respiratory tract pathogens and intranasal vaccination. This PhD project investigated the frequencies of TFH in human NALT and PBMC in children and adults. The effects of CpG-DNA and live attenuated influenza vaccine (LAIV) on TFH in human NALT and the TFH-mediated B cell immunity to influenza virus were studied. The importance of the cytokine IL-21 and plasmacytoid dendritic cells (pDC) in TFH cell-mediated B cell antibody production was also investigated. Adenotonsillar MNC and PBMC were isolated from adenotonsillar tissues and peripheral blood respectively. TFH (CD4+ CXCR5high ICOShigh) numbers and function were analysed by flowcytometry and intracellular cytokine staining. Purified TFH (CD4+ CXCR5hi) and non-TFH cells (CD4+ CXCR5-) were co-cultured with B cells in the presence of influenza virus antigen and CpG-DNA or of LAIV. Purified pDC were added to the TFH-B cell co-culture to study their importance in TFH -mediated B cell antibody production. Haemagglutinin (HA)-specific antibody production was analysed by ELISA and ELISpot assay. IL-21 receptor blocking by neutralization was used to study the importance of IL-21 in TFH-mediated B cell antibody production. A prominent number of TFH were found in human NALT which were considerably higher than in PBMC. There was an age-associated difference in TFH numbers in NALT and BPMC, i.e. the mean TFH number was higher in children than in adults. TFH in NALT were shown to express high levels of IL-4, IL-10 and IL-21 and that were important for B cell antibody production. A good correlation between the numbers of GC B cell and TFH in NALT was seen. Co-culture of purified TFH but not non-TFH with B cells promoted antibody production. Stimulation of adenotonsillar MNC by CpG-DNA significantly increased TFH number and that was correlated with HA-specific antibody production following influenza antigen stimulation. Co-incubation of TFH-B cell with pDC enhanced the CpG-DNA-mediated antibody production. We also found that stimulation with LAIV significantly increased TFH number and that was correlated with HA-specific antibody production. Blocking the IL-21R significantly reduced the number of TFH that was correlated with a significant reduction of HA-specific antibody production. Enhancing vaccine immunogenicity through modulation of TFH numbers or function in human NALT using immunological adjuvants such as CpG-DNA and through intranasal vaccination may be an effective vaccination strategy against respiratory pathogens

Activation and Induction of Antigen-Specific T Follicular Helper Cells Play a Critical Role in Live-Attenuated Influenza Vaccine-Induced Human Mucosal Anti-influenza Antibody Response

There is increasing interest recently in developing intranasal vaccines against respiratory tract infections. The antibody response is critical for vaccine-induced protection, and T follicular helper cells (TFH) are considered important for mediating the antibody response. Most data supporting the role for TFH in the antibody response are from animal studies, and direct evidence from humans is limited, apart from the presence of TFH-like cells in blood. We studied the activation and induction of TFH and their role in the anti-influenza antibody response induced by a live-attenuated influenza vaccine (LAIV) in human nasopharynx-associated lymphoid tissue (NALT). TFH activation in adenotonsillar tissues was analyzed by flow cytometry, and anti-hemagglutinin (anti-HA) antibodies were examined following LAIV stimulation of tonsillar mononuclear cells (MNC). Induction of antigen-specific TFH by LAIV was studied by flow cytometry analysis of induced TFH and CD154 expression. LAIV induced TFH proliferation, which correlated with anti-HA antibody production, and TFH were shown to be critical for the antibody response. Induction of TFH from naive T cells by LAIV was shown in newly induced TFH expressing BCL6 and CD21, followed by the detection of anti-HA antibodies. Antigen specificity of LAIV-induced TFH was demonstrated by expression of the antigen-specific T cell activation marker CD154 upon challenge by H1N1 virus antigen or HA. LAIV-induced TFH differentiation was inhibited by BCL6, interleukin-21 (IL-21), ICOS, and CD40 signaling blocking, and that diminished anti-HA antibody production. In conclusion, we demonstrated the induction by LAIV of antigen-specific TFH in human NALT that provide critical support for the anti-influenza antibody response. Promoting antigen-specific TFH in NALT by use of intranasal vaccines may provide an effective vaccination strategy against respiratory infections in humans. IMPORTANCE Airway infections, such as influenza, are common in humans. Intranasal vaccination has been considered a biologically relevant and effective way of immunization against airway infection. The vaccine-induced antibody response is crucial for protection against infection. Recent data from animal studies suggest that one type of T cells, TFH, are important for the antibody response. However, data on whether TFH-mediated help for antibody production operates in humans are limited due to the lack of access to human immune tissue containing TFH. In this study, we demonstrate the induction of TFH in human immune tissue, providing critical support for the anti-influenza antibody response, by use of an intranasal influenza vaccine. Our findings provide direct evidence that TFH play a critical role in vaccine-induced immunity in humans and suggest a novel strategy for promoting such cells by use of intranasal vaccines against respiratory infections

Seroprevalence of antibodies to SARS-CoV-2 among blood donors in the early month of the pandemic in Saudi Arabia

Background: Serologic testing provides better understanding of SARS-CoV-2 prevalence and its transmission. This study was an investigation of the prevalence of antibodies to SARS-CoV-2 among blood donors in Saudi Arabia. Objective: To estimate the seroprevalence of anti-SARS-CoV-2 antibodies among blood donors in Saudi Arabia during the early phase of the COVID-19 pandemic. Methods: Serology results and epidemiological data were analyzed for 837 adult blood donors, with no confirmed SARS-CoV-2 infection, in Saudi Arabia from 20th to 25th May 2020. Seroprevalence was determined using electrochemical immunoassay to detect anti-SARS-CoV-2 antibodies. Results: The overall seroprevalence of anti-SARS-CoV-2 antibodies was 1.4% (12/837). Non-citizens had higher seroprevalence compared with citizens (OR 13.6, p = 0.001). Secondary education was significantly associated with higher seroprevalence compared with higher education (OR 6.8, p = 0.005). The data showed that the highest seroprevalence was in Makkah (8.1%). Uisng Makkah seroprevalence as the reference, the seroprevalence in other areas was: Madinah 4.1% (OR 0.48, 95% CI 0.12-1.94), Jeddah 2.3% (OR 0.27, 95% CI 0.31-2.25), and Qassim 2.9 % (OR 0.34, 95% CI 0.04-2.89) and these were not statistically different from seroprevalence in the Makkah region. Conclusions: At the early months of the COVID-19 pandemic in Saudi Arabia, the seroprevalence of antibodies to SARS-CoV-2 among blood donors was low, but was higher among non-citizens. These findings may indicate that non-citizens and less educated individuals may be less attentive to preventive measures. Monitoring seroprevalence trends over time require repeated sampling

Household Air Pollution Causes Dose-dependent Inflammation and Altered Phagocytosis in Human Macrophages

Background Three billion people are exposed to household air pollution from biomass fuel use. Exposure is associated with higher incidence of pneumonia, and possibly tuberculosis. Understanding mechanisms underlying these defects would improve preventive strategies. Methods We used human alveolar macrophages obtained from healthy Malawian adults exposed naturally to household air pollution, and compared with human monocyte-derived macrophages exposed in vitro to respirable-sized particulates. Cellular inflammatory response was assessed by: IL-6 and IL-8 production in response to particulate challenge; phagocytosis of fluorescent-labelled beads and intraphagosomal oxidative burst capacity; ingestion and killing of Streptococcus pneumoniae and Mycobacterium tuberculosis measured by microscopy and quantitative culture. Particulate ingestion was quantified by digital image analysis. Results We were able to reproduce the carbon loading of naturally exposed alveolar macrophages by in vitro exposure of monocyte derived macrophages. Fine carbon black induced IL-8 release from monocyte derived and alveolar macrophages (p<0.05), with similar magnitude responses (log10 increases of 0.93 [SEM 0.2] vs 0.74 [SEM 0.19] respectively). Phagocytosis of pneumococci and mycobacteria was impaired with higher particulate loading. High particulate loading corresponded with a lower oxidative burst capacity (p=0.0015). There was no overall effect on killing of M. tuberculosis. Conclusion Alveolar macrophage function is altered by particulate loading. Our macrophage model is comparable morphologically to the in vivo uptake of particulates. Wood smoke exposed cells demonstrate reduced phagocytosis but unaffected mycobacterial killing, suggesting defects related to chronic wood smoke inhalation limited to specific innate immune functions

SARS-CoV-2 vaccination modelling for safe surgery to save lives: data from an international prospective cohort study

Background: Preoperative SARS-CoV-2 vaccination could support safer elective surgery. Vaccine numbers are limited so this study aimed to inform their prioritization by modelling. Methods: The primary outcome was the number needed to vaccinate (NNV) to prevent one COVID-19-related death in 1 year. NNVs were based on postoperative SARS-CoV-2 rates and mortality in an international cohort study (surgical patients), and community SARS-CoV-2 incidence and case fatality data (general population). NNV estimates were stratified by age (18-49, 50-69, 70 or more years) and type of surgery. Best- and worst-case scenarios were used to describe uncertainty. Results: NNVs were more favourable in surgical patients than the general population. The most favourable NNVs were in patients aged 70 years or more needing cancer surgery (351; best case 196, worst case 816) or non-cancer surgery (733; best case 407, worst case 1664). Both exceeded the NNV in the general population (1840; best case 1196, worst case 3066). NNVs for surgical patients remained favourable at a range of SARS-CoV-2 incidence rates in sensitivity analysis modelling. Globally, prioritizing preoperative vaccination of patients needing elective surgery ahead of the general population could prevent an additional 58 687 (best case 115 007, worst case 20 177) COVID-19-related deaths in 1 year. Conclusion: As global roll out of SARS-CoV-2 vaccination proceeds, patients needing elective surgery should be prioritized ahead of the general population