Combining SARS-CoV-2 interferon-gamma release assay with humoral response assessment to define immune memory profiles.

OBJECTIVES: In the post-SARS-CoV-2 pandemic era, "breakthrough infections" are still documented, due to variants of concerns (VoCs) emergence and waning humoral immunity. Despite widespread utilization, the definition of the anti-Spike (S) immunoglobulin-G (IgG) threshold to define protection has unveiled several limitations. Here, we explore the advantages of incorporating T-cell response assessment to enhance the definition of immune memory profile. METHODS: SARS-CoV-2 interferon-gamma release assay test (IGRA) was performed on samples collected longitudinally from immunocompetent healthcare workers throughout their immunization by infection and/or vaccination, anti-receptor-binding domain IgG levels were assessed in parallel. The risk of symptomatic infection according to cellular/humoral immune capacities during Omicron BA.1 wave was then estimated. RESULTS: Close to 40% of our samples were exclusively IGRA-positive, largely due to time elapsed since their last immunization. This suggests that individuals have sustained long-lasting cellular immunity, while they would have been classified as lacking protective immunity based solely on IgG threshold. Moreover, the Cox regression model highlighted that Omicron BA.1 circulation raises the risk of symptomatic infection while increased anti-receptor-binding domain IgG and IGRA levels tended to reduce it. CONCLUSION: The discrepancy between humoral and cellular responses highlights the significance of assessing the overall adaptive immune response. This integrated approach allows the identification of vulnerable subjects and can be of interest to guide antiviral prophylaxis at an individual level.

Prior SARS-CoV-2 infection enhances and reshapes spike protein-specific memory induced by vaccination.

The diversity of vaccination modalities and infection history are both variables that have an impact on the immune memory of individuals vaccinated against SARS-CoV-2. To gain more accurate knowledge of how these parameters imprint on immune memory, we conducted a long-term follow-up of SARS-CoV-2 spike protein-specific immune memory in unvaccinated and vaccinated COVID-19 convalescent individuals as well as in infection-naïve vaccinated individuals. Here, we report that individuals from the convalescent vaccinated (hybrid immunity) group have the highest concentrations of spike protein-specific antibodies at 6 months after vaccination. As compared with infection-naïve vaccinated individuals, they also display increased frequencies of an atypical mucosa-targeted memory B cell subset. These individuals also exhibited enhanced TH1 polarization of their SARS-CoV-2 spike protein-specific follicular T helper cell pool. Together, our data suggest that prior SARS-CoV-2 infection increases the titers of SARS-CoV-2 spike protein-specific antibody responses elicited by subsequent vaccination and induces modifications in the composition of the spike protein-specific memory B cell pool that are compatible with enhanced functional protection at mucosal sites.

Dynamics of viral shedding during ancestral or Omicron BA.1 SARS-CoV-2 infection and enhancement of pre-existing immunity during breakthrough infections.

Omicron variant is circulating in the presence of a globally acquired immunity unlike the ancestral SARS-CoV-2 isolate. Herein, we investigated the normalized viral load dynamics and viral culture status in 44 fully vaccinated healthcare workers (HCWs) infected with the Omicron BA.1 variant. Viral load dynamics of 38 unvaccinated HCWs infected with the 20A variant during the first pandemic wave was also studied. We then explored the impact of Omicron infection on pre-existing immunity assessing anti-RBD IgG levels, neutralizing antibody titres against 19A, Delta and Omicron isolates, as well as IFN-γ release following cell stimulation with SARS-CoV-2 peptides. We reported that two weeks after diagnosis a greater proportion of HCWs infected with 20A (78.9%, 15/19) than with Omicron BA.1 (44.7%, 17/38; p = 0.02) were still positive by RT-qPCR. We found that Omicron breakthrough infections led to an overall enhancement of vaccine-induced humoral and cellular immunity as soon as a median [interquartile range] of 8 [7-9] days post symptom onset. Among samples with similar high viral loads, non-culturable samples exhibited higher neutralizing antibody titres and anti-RBD IgG levels than culturable samples. Additionally, Omicron infection led to an enhancement of antibodies neutralization capacity against other SARS-CoV-2 isolates. Taken together, the results suggest that Omicron BA.1 vaccine breakthrough infection is associated with a faster viral clearance than that of the ancestral SARS-CoV-2, in addition this new variant leads to a rapid enhancement of the humoral response against multiple SARS-CoV-2 variants, and of the cellular response.

Specific detection of memory T-cells in COVID-19 patients using standardized whole-blood Interferon gammarelease assay.

Antigen-specific T-cells are essential for protective immunity against SARS-CoV-2. We set up a semi-automated whole-blood Interferon-gamma release assay (WB IGRA) to monitor the T-cell response after stimulation with SARS-CoV-2 peptide pools. We report that the WB IGRA is complementary to serological assays to assess SARS-CoV-2 immunity.

Formulation of HIV-1 Tat and p24 antigens by PLA nanoparticles or MF59 impacts the breadth, but not the magnitude, of serum and faecal antibody responses in rabbits.

Biodegradable nanoparticles coated with proteins represent a promising method for in vivo delivery of vaccines. Here we used a rabbit model to compare quantitatively and qualitatively the antibody responses induced by poly(D,L-lactide) nanoparticles (PLA) and by emulsion adjuvant MF59 using three HIV-1 antigens: p24gag, WT Tat and a mutated, detoxified form of Tat. We could show that all antigens and adjuvants lead to the induction of similar level of IgG titres in serum when injected subcutaneously. p24, but not Tat, could also induce faecal IgG in rabbits when formulated with PLA or MF59. The nature of the adjuvant had consequences on the spectrum of specificity induced, depending on the antigen: PLA adjuvant focussed the anti-p24 response to an immunodominant domain when compared to MF59. With wild-type Tat, no difference between adjuvants was observed in the spectrum of specificity induced. On the opposite, detoxified Tat coated on PLA increased the number of epitopes recognized by serum IgG compared to MF59 adjuvantation. The impact of these qualitative differences depending on the antigen/adjuvant associations will be important to take into account for further designs of vaccinal formulation using particulate adjuvants.

Coadsorption of HIV-1 p24 and gp120 proteins to surfactant-free anionic PLA nanoparticles preserves antigenicity and immunogenicity.

Biodegradable micro- or nanoparticles with surface adsorbed antigens represent a promising method for in vivo delivery of vaccines. Most vaccines, licensed or under development, are based on combined delivery of multiple antigens. Thus, we investigated the feasibility of combining two vaccine antigens, HIV-1 p24 and gp120 proteins, on the surface of surfactant-free anionic PLA nanoparticles obtained by an improved solvent diffusion method. The analysis of adsorption isotherms has shown that both proteins had similar and high affinities for the nanoparticles. Coadsorption of p24 and gp120 onto the same PLA particle was evidenced by sandwich ELISA, using antibodies directed against one protein for particle capture and the other one for detection. To assess structural integrity, the antigenicity of free and PLA-adsorbed antigens was compared by competition ELISA, using a set of 6 anti-p24 and 7 anti-gp120 antibodies, as well as soluble CD4. The antigenicity of proteins on the nanoparticle surface was well preserved, adsorbed either individually or in combination. Furthermore, both antigens maintained their immunogenicity, since high antibody titres (10(6) for p24 and 10(5) for gp120) were elicited in mice with monovalent and divalent PLA formulations. Taken together our results show that development of multivalent vaccines based on anionic PLA nanoparticles is possible. Moreover, coadsorption of a ligand for cell-specific targeting or of an immunostimulatory molecule will further extend the field of application of delivery systems based on charged micro- and nanoparticles.

Genome-wide analysis of gene expression in neuroblastomas detected by mass screening.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and the third most common pediatric cancer. Although numerous factors including patient age, disease stage and genetic abnormalities have been shown to be predictive of outcome, the mechanisms responsible for the highly variable clinical behavior of this tumor remain largely unknown. In order to gain new insights into the biology of this tumor, we performed microarray analysis and compared the gene expression patterns of NB detected by mass screening, characterized by highly probable spontaneous regression, versus stage 4 NB with poor prognosis. The bioinformatics analysis revealed a set of 19 discriminatory genes that may play a significant role in the natural progression of the disease. Validation of these results and further mechanistic studies would shed new light on the biology of tumor progression, and provide new tools to predict clinical outcome in children with NB.