SARS-CoV-2 Vaccine Responses in Individuals with Antibody Deficiency: Findings from the COV-AD Study

Background

Vaccination prevents severe morbidity and mortality from COVID-19 in the general population. The immunogenicity and efficacy of SARS-CoV-2 vaccines in patients with antibody deficiency is poorly understood.

Objectives

COVID-19 in patients with antibody deficiency (COV-AD) is a multi-site UK study that aims to determine the immune response to SARS-CoV-2 infection and vaccination in patients with primary or secondary antibody deficiency, a population that suffers from severe and recurrent infection and does not respond well to vaccination.

Methods

Individuals on immunoglobulin replacement therapy or with an IgG less than 4 g/L receiving antibiotic prophylaxis were recruited from April 2021. Serological and cellular responses were determined using ELISA, live-virus neutralisation and interferon gamma release assays. SARS-CoV-2 infection and clearance were determined by PCR from serial nasopharyngeal swabs.

Results

A total of 5.6% (n?=?320) of the cohort reported prior SARS-CoV-2 infection, but only 0.3% remained PCR positive on study entry. Seropositivity, following two doses of SARS-CoV-2 vaccination, was 54.8% (n?=?168) compared with 100% of healthy controls (n?=?205). The magnitude of the antibody response and its neutralising capacity were both significantly reduced compared to controls. Participants vaccinated with the Pfizer/BioNTech vaccine were more likely to be seropositive (65.7% vs. 48.0%, p?=?0.03) and have higher antibody levels compared with the AstraZeneca vaccine (IgGAM ratio 3.73 vs. 2.39, p?=?0.0003). T cell responses post vaccination was demonstrable in 46.2% of participants and were associated with better antibody responses but there was no difference between the two vaccines. Eleven vaccine-breakthrough infections have occurred to date, 10 of them in recipients of the AstraZeneca vaccine.

Conclusion

SARS-CoV-2 vaccines demonstrate reduced immunogenicity in patients with antibody deficiency with evidence of vaccine breakthrough infection.

     

Overexpression and potential targeting of the oncofoetal antigen 5T4 in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is resistant to conventional treatments. Novel, targeted treatments are hampered by the relative lack of MPM-associated tumour antigens. The aim of this study was to evaluate the level of expression and the relevance of 5T4 as a tumour-associated antigen in MPM. 5T4 expression was assessed by Western blotting, flow cytometry, immuno-cytochemistry and -histochemistry in 11 mesothelioma cell lines, 21 tumour biopsies, and ex vivo tumour cells obtained from the pleural fluid (PF) of 10 patients. 5T4 antibody levels were also determined in the plasma of patients and healthy donors. The susceptibility of MPM cells to 5T4-specific T-cell-mediated killing was determined using an HLA-A2(+), CD8(+) T-cell line, developed against the 5T4(17-25) peptide. We report here that cell surface 5T4 expression was detected in all mesothelioma cell lines and PF cell samples. Mesothelin and CD200, a suggested mesothelioma marker, were co-expressed with 5T4 on tumour cells in PF. Immunohistochemistry confirmed overexpression of 5T4, similar to mesothelin, on tumour cells but not on reactive stroma in all tissue sections tested. Median 5T4 antibody levels were 46% higher in patient than in healthy donor plasma, indicating immune recognition. Importantly, 5T4-specific CD8(+) T-cells were able to kill four out of six HLA-A2(+) MPM cell lines but not an HLA-A2(-) cell line, demonstrating immune recognition of MPM-associated 5T4 antigen at the effector T-cell level. We conclude that 5T4 is a potential new antigen for targeted therapies such as immunotherapy in MPM, as it is overexpressed on mesothelioma cells and recognised by 5T4-specific cytotoxic T-cells. Our findings have been translated into a Phase II clinical trial applying 5T4-targeted therapies in MPM patients.     

Effects of Na+/H+ exchanger inhibitors on subcellular localisation of endocytic organelles and intracellular dynamics of protein transduction domains HIV-TAT peptide and octaarginine.

Protein transduction domains such as those derived from the HIV protein TAT have great potential as vectors for delivery of therapeutic entities such as genes and proteins into cells. Extensive studies have shown that a major fraction of the most studied variants enters cells via an endocytic mechanism. However, controversy surrounds the exact uptake mechanism and whether a specific pathway is utilised. Studies showing inhibition of uptake of protein transduction domains in the presence of ion-transport inhibitors such as amiloride and its more potent analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA) suggest a link between peptide internalisation and macropinocytosis. In this study, using immunolabelling of early and late components of the endocytic pathway, we show that treatment of cells with EIPA and to a lesser extent amiloride affects the morphology and subcellular location of early, late endosomes and lysosomes. Enlarged early and late endocytic structures were observed in EIPA-treated cells, and these organelles accumulated in a perinuclear region. Results from experiments investigating the effects of EIPA on distribution of fluorescent octaarginine were in agreement with the immunolocalisation studies. Treatment of the CD34(+) leukaemia cell line KG1a with EIPA in the presence of fluorescent conjugates of HIV-TAT peptide and octaarginine showed distinct vesicular staining in agreement with untreated cells but EIPA-treated cells were additionally characterized by increased localization of the peptides in the cytosol. At levels previously shown to inhibit uptake of HIV-TAT peptide and octaarginine in other cell lines, EIPA was without major effect on uptake of both peptides in KG1a cells.