A FluoroSpot B assay for the detection of IgA and IgG SARS-CoV-2 spike-specific memory B cells: Optimization and qualification for use in COVID-19 vaccine trials.

BACKGROUND: The generation of antigen-specific memory B cells is crucial to the long-term effectiveness of vaccines. When the protective antibodies circulating in the blood wane, memory B cells (MBC) can be rapidly reactivated and differentiated into antibody-secreting cells during a new infection. Such MBC responses are considered to be key in providing long-term protection after infection or vaccination. Here, we describe the optimization and qualification of a FluoroSpot assay to measure MBCs directed against the SARS-CoV-2 spike protein in the peripheral blood, for use in COVID-19 vaccine trials. METHODS: We developed a FluoroSpot assay enabling simultaneous enumeration of B cells secreting IgA or IgG spike-specific antibodies after polyclonal stimulation of peripheral blood mononuclear cells (PBMCs) with interleukin-2 and the toll-like receptor agonist R848 for 5 days. The antigen coating was optimized using a capture antibody directed against the spike subunit-2 glycoprotein of SARS-CoV-2 to immobilize recombinant trimeric spike protein onto the membrane. RESULTS: Compared to a direct spike protein coating, the addition of a capture antibody increased the number and the quality of detected spots for both spike-specific IgA and IgG secreting cells in PBMCs from COVID-19 convalescents. The qualification showed good sensitivity of the dual-color IgA-IgG FluoroSpot assay, with lower limits of quantitation of 18 background-subtracted (BS) antibody-secreting cells (ASCs)/well for spike-specific IgA and IgG responses. Linearity was demonstrated at values ranging from 18 to 73 and from 18 to 607 BS ASCs/well for spike-specific IgA and IgG, respectively, as was precision, with intermediate precision (percentage geometric coefficients of variation) of 12% and 26% for the proportion of spike-specific IgA and IgG MBCs (ratio specific/total IgA or Ig). The assay was specific, since no spike-specific MBCs were detected in PBMCs from pre-pandemic samples; the results were below the limit of detection of 17 BS ASCs/well. CONCLUSIONS: These results show that the dual-color IgA-IgG FluoroSpot provides a sensitive, specific, linear, and precise tool to detect spike-specific MBC responses. This MBC FluoroSpot assay is a method of choice for monitoring spike-specific IgA and IgG MBC responses induced by COVID-19 candidate vaccines in clinical trials.

A novel vaccine adjuvant based on straight polyacrylate potentiates vaccine-induced humoral and cellular immunity in cynomolgus macaques.

Adjuvants are central to the efficacy of subunit vaccines. Although several new adjuvants have been approved in human vaccines over the last decade, the panel of adjuvants in licensed human vaccines remains small. There is still a need for novel adjuvants that can be safely used in humans, easy to source and to formulate with a wide range of antigens and would be broadly applicable to a wide range of vaccines. In this article, using the Respiratory Syncytial Virus (RSV) nanoparticulate prefusion F model antigen developed by Sanofi, we demonstrate in the macaque model that the polyacrylate (PAA)-based adjuvant SPA09 is well tolerated and increases vaccine antigen-specific humoral immunity (sustained neutralizing antibodies, memory B cells and mucosal immunity) and elicits strong TH1-type responses (based on IFNγ and IL-2 ELISpots) in a dose-dependent manner. These data warrant further development of the SPA09 adjuvant for evaluation in clinical trials.

Nine µg intradermal influenza vaccine and 15 µg intramuscular influenza vaccine induce similar cellular and humoral immune responses in adults.

Intanza® 9 µg (Sanofi Pasteur), a trivalent split-virion vaccine administered by intradermal (ID) injection, was approved in Europe in 2009 for the prevention of seasonal influenza in adults 18 to 59 years. Here, we examined the immune responses induced in adults by the ID 9 µg vaccine and the standard trivalent intramuscular (IM) vaccine (Vaxigrip® 15 µg, Sanofi Pasteur). This trial was a randomized, controlled, single-center, open-label study in healthy adults 18 to 40 years of age during the 2007/8 influenza season. Subjects received a single vaccination with the ID 9 µg (n=38) or IM 15 µg (n=42) vaccine. Serum, saliva, and peripheral blood mononuclear cells were collected up to 180 days post-vaccination. Geometric mean hemagglutination inhibition titers, seroprotection rates, seroconversion rates, and pre-vaccination-to-post-vaccination ratios of geometric mean hemagglutination inhibition titers did not differ between the two vaccines. Compared with pre-vaccination, the vaccines induced similar increases in vaccine-specific circulating B cells at day 7 but did not induce significant increases in vaccine-specific memory B cells at day 180. Cell-mediated immunity to all three vaccine strains, measured in peripheral blood mononuclear cells, was high at baseline and not increased by either vaccine. Neither vaccine induced a mucosal immune response. These results show that the humoral and cellular immune responses to the ID 9 µg vaccine are similar to those to the standard IM 15 µg vaccine.

HLA-DR and HLA-DP restricted epitopes from human cytomegalovirus glycoprotein B recognized by CD4+ T-cell clones from chronically infected individuals.

PURPOSE: Helper CD4(+) T cells presumably play a major role in controlling cytomegalovirus (CMV) by providing help to specific B and CD8(+) cytotoxic T cells, as well as through cytotoxicity-mediated mechanisms. Since CMV glycoprotein B (gB) is a major candidate for a subunit vaccine against CMV, we searched for gB-epitopes presented by human leukocyte antigen (HLA)-class II molecules. METHODS: Dendritic cells obtained from CMV-seropositive donors were loaded with a recombinant gB and co-cultured with autologous CD4(+) T cells. Microcultures that specifically recognized gB were cloned by limiting dilution using autologous Epstein-Barr virus (EBV)-immortalized B cells pulsed with gB as antigen-presenting cells. To pinpoint precisely the region encoding the natural epitope recognized by a given CD4(+) clone, we assessed the recognition of recombinant Escherichia coli expressing gB-overlapping polypeptides after their processing by autologous EBV-B cells. RESULTS: We isolated several gB-specific CD4(+) T-cell clones directed against peptides gB(190-204), gB(396-410), gB(22-36) and gB(598-617) presented by HLA-DR7, HLA-DP10 and HLA-DP2. While their precise role in controlling CMV infection remains to be established, gB-specific CD4(+) T cells are likely to act by directly targeting infected HLA-class II cells in vivo, as suggested by their recognition of EBV-B cells infected by the Towne CMV strain. CONCLUSIONS: The characterization of such gB-epitopes presented by HLA-class II should help to understand the contribution of CD4(+) T-cell responses to CMV and may be of importance both in designing a vaccine against CMV infection and in immunomonitoring of subjects immunized with recombinant gB or with vectors encoding gB.