Long-term recruitment of peripheral immune cells to brain scars after a neonatal insult.

Although brain scars in adults have been extensively studied, there is less data available regarding scar formation during the neonatal period, and the involvement of peripheral immune cells in this process remains unexplored in neonates. Using a murine model of neonatal hypoxic-ischemic encephalopathy (HIE) and confocal microscopy, we characterized the scarring process and examined the recruitment of peripheral immune cells to cortical and hippocampal scars for up to 1 year post-insult. Regional differences in scar formation were observed, including the presence of reticular fibrotic networks in the cortex and perivascular fibrosis in the hippocampus. We identified chemokines with chronically elevated levels in both regions and demonstrated, through a parabiosis-based strategy, the recruitment of lymphocytes, neutrophils, and monocyte-derived macrophages to the scars several weeks after the neonatal insult. After 1 year, however, neutrophils and lymphocytes were absent from the scars. Our data indicate that peripheral immune cells are transient components of HIE-induced brain scars, opening up new possibilities for late therapeutic interventions.

From a recombinant key antigen to an accurate, affordable serological test: Lessons learnt from COVID-19 for future pandemics.

Serological tests detect antibodies generated by infection or vaccination, and are indispensable tools along different phases of a pandemic, from early monitoring of pathogen spread up to seroepidemiological studies supporting immunization policies. This work discusses the development of an accurate and affordable COVID-19 antibody test, from production of a recombinant protein antigen up to test validation and economic analysis. We first developed a cost-effective, scalable technology to produce SARS-COV-2 spike protein and then used this antigen to develop an enzyme-linked immunosorbent assay (ELISA). A receiver operator characteristic (ROC) analysis allowed optimizing the cut-off and confirmed the high accuracy of the test: 98.6% specificity and 95% sensitivity for 11+ days after symptoms onset. We further showed that dried blood spots collected by finger pricking on simple test strips could replace conventional plasma/serum samples. A cost estimate was performed and revealed a final retail price in the range of one US dollar, reflecting the low cost of the ELISA test platform and the elimination of the need for venous blood sampling and refrigerated sample handling in clinical laboratories. The presented workflow can be completed in 4 months from first antigen expression to final test validation. It can be applied to other pathogens and in future pandemics, facilitating reliable and affordable seroepidemiological surveillance also in remote areas and in low-income countries.

The sequences encoded by immunoglobulin diversity (DH ) gene segments play key roles in controlling B-cell development, antigen-binding site diversity, and antibody production.

Although at first glance the diversity of the immunoglobulin repertoire appears random, there are a number of mechanisms that act to constrain diversity. For example, key mechanisms controlling the diversity of the third complementarity determining region of the immunoglobulin heavy chain (CDR-H3) include natural selection of germline diversity (DH ) gene segment sequence and somatic selection upon passage through successive B-cell developmental checkpoints. To test the role of DH gene segment sequence, we generated a panel of mice limited to the use of a single germline or frameshifted DH gene segment. Specific individual amino acids within core DH gene segment sequence heavily influenced the absolute numbers of developing and mature B-cell subsets, antibody production, epitope recognition, protection against pathogen challenge, and susceptibility to the production of autoreactive antibodies. At the tip of the antigen-binding loop (PDB position 101) in CDR-H3, both natural (germline) and somatic selection favored tyrosine while disfavoring the presence of hydrophobic amino acids. Enrichment for arginine in CDR-H3 appeared to broaden recognition of epitopes of varying hydrophobicity, but led to diminished binding intensity and an increased likelihood of generating potentially pathogenic dsDNA-binding autoreactive antibodies. The phenotype of altering the sequence of the DH was recessive for T-independent antibody production, but dominant for T-cell-dependent responses. Our work suggests that the antibody repertoire is structured, with the sequence of individual DH selected by evolution to preferentially generate an apparently preferred category of antigen-binding sites. The result of this structured approach appears to be a repertoire that has been adapted, or optimized, to produce protective antibodies for a wide range of pathogen epitopes while reducing the likelihood of generating autoreactive specificities.

Genetic Evidence and Host Immune Response in Persons Reinfected with SARS-CoV-2, Brazil.

The dynamics underlying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection remain poorly understood. We identified a small cluster of patients in Brazil who experienced 2 episodes of coronavirus disease (COVID-19) in March and late May 2020. In the first episode, patients manifested an enhanced innate response compared with healthy persons, but neutralizing humoral immunity was not fully achieved. The second episode was associated with different SARS-CoV-2 strains, higher viral loads, and clinical symptoms. Our finding that persons with mild COVID-19 may have controlled SARS-CoV-2 replication without developing detectable humoral immunity suggests that reinfection is more frequent than supposed, but this hypothesis is not well documented.

Protective effect of fungal extracellular vesicles against murine candidiasis.

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL-12p70, TGFß, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.

Immunomodulating role of IL-10-producing B cells in Leishmania amazonensis infection.

This work aims to study the immunomodulation of B lymphocytes during L. amazonensis infection. We demonstrated in this study that follicular B cells from draining lymph nodes of infected wild type BALB/c mice are the major source of IL-10 during infection. We infected BALB/Xid mice that developed smaller lesions in comparison with the control, but the parasite load obtained from the infected tissues was similar in both groups. We observed a reduction in the number of follicular B cells from BALB/Xid mice in relation to WT mice and, consequently, lower levels of IgM, IgG, IgG1, IgG2a and IgG2b in the serum of BALB/Xid when compared with wild type mice. BALB/Xid mice also presented lower levels of IL-10 in the infected footpad, draining lymph nodes and in the spleen when compared with WT infected tissues. We did not detect differences in the number of IL-10 producing CD4+ and CD8+ T cells between WT and BALB/Xid mice; however, a strong reduction of IL-10 producing follicular B cells was noted in BALB/Xid mice. When analyzed together, our data indicate that B cells are related with lesion pathogenesis through the production of antibodies and IL-10.

Long-term maintenance of polysaccharide-specific antibodies by IgM-secreting cells.

Many bacteria-associated polysaccharides induce long-lived Ab responses that protect against pathogenic microorganisms. The maintenance of polysaccharide-specific Ab titers may be due to long-lived plasma cells or ongoing Ag-driven B cell activation due to polysaccharide persistence. BALB/c and V(H)J558.3 transgenic mice respond to α1→3-dextran (DEX) by generating a peak anti-DEX response at 7 d, followed by maintenance of serum Ab levels for up to 150 d. Analysis of the cellular response to DEX identified a population of short-lived, cyclophosphamide-sensitive DEX-specific plasmablasts in the spleen, and a quiescent, cyclophosphamide-resistant DEX-specific Ab-secreting population in the bone marrow. BrdU pulse-chase experiments demonstrated the longevity of the DEX-specific Ab-secreting population in the bone marrow. Splenic DEX-specific plasmablasts were located in the red pulp with persisting DEX-associated CD11c(+) dendritic cells 90 d after immunization, whereas DEX was not detected in the bone marrow after 28 d. Selective depletion of short-lived DEX-specific plasmablasts and memory B1b B cells using cyclophosphamide and anti-CD20 treatment had a minimal impact on the maintenance of serum anti-DEX Abs. Collectively, these findings demonstrate that the maintenance of serum polysaccharide-specific Abs is the result of continuous Ag-driven formation of short-lived plasmablasts in the spleen and a quiescent population of Ab-secreting cells maintained in the bone marrow for a long duration.

Oligosymptomatic long-term carriers of SARS-CoV-2 display impaired innate resistance but increased high-affinity anti-spike antibodies.

The vast spectrum of clinical features of COVID-19 keeps challenging scientists and clinicians. Low resistance to infection might result in long-term viral persistence, but the underlying mechanisms remain unclear. Here, we studied the immune response of immunocompetent COVID-19 patients with prolonged SARS-CoV-2 infection by immunophenotyping, cytokine and serological analysis. Despite viral loads and symptoms comparable to regular mildly symptomatic patients, long-term carriers displayed weaker systemic IFN-I responses and fewer circulating pDCs and NK cells at disease onset. Type 1 cytokines remained low, while type-3 cytokines were in turn enhanced. Of interest, we observed no defects in antigen-specific cytotoxic T cell responses, and circulating antibodies displayed higher affinity against different variants of SARS-CoV-2 Spike protein in these patients. The identification of distinct immune responses in long-term carriers adds up to our understanding of essential host protective mechanisms to ensure tissue damage control despite prolonged viral infection.

The peritoneal cavity B-2 antibody repertoire appears to reflect many of the same selective pressures that shape the B-1a and B-1b repertoires.

To assess the extent and nature of somatic categorical selection of CDR-3 of the Ig H chain (CDR-H3) content in peritoneal cavity (PerC) B cells, we analyzed the composition of V(H)7183DJCµ transcripts derived from sorted PerC B-1a, B-1b, and B-2 cells. We divided these sequences into those that contained N nucleotides (N(+)) and those that did not (N(-)) and then compared them with sequences cloned from sorted IgM(+)IgD(+) B cells from neonatal liver and both wild-type and TdT-deficient adult bone marrow. We found that the PerC B-1a N(-) repertoire is enriched for the signatures of CDR-H3 sequences present in neonatal liver and shares many features with the B-1b N(-) repertoire, whereas the PerC B-1a N(+), B-1b N(+), and B-2 N(+) repertoires are enriched for adult bone marrow sequence signatures. However, we also found several sequence signatures that were not shared with other mature perinatal or adult B cell subsets but were either unique or variably shared between the two or even among all three of the PerC subsets that we examined. These signatures included more sequences lacking N nucleotides in the B-2 population and an increased use of D(H) reading frame 2, which created CDR-H3s of greater average hydrophobicity. These findings provide support for both ontogenetic origin and shared Ag receptor-influenced selection as the mechanisms that shape the unique composition of the B-1a, B-1b, and B-2 repertoires. The PerC may thus serve as a general reservoir for B cells with Ag binding specificities that are uncommon in other mature compartments.

The CDR-H3 repertoire from TdT-deficient adult bone marrow is a close, but not exact, homologue of the CDR-H3 repertoire from perinatal liver.

Compared with adult bone marrow (BM), the composition of the perinatal liver CDR-3 of the Ig H chain (CDR-H3) repertoire is marked by a paucity of N nucleotides and by enrichment for use of J(H) proximal DQ52 and D(H) proximal V(H) and J(H) gene segments. To test the extent to which these differences reflect limited perinatal TdT activity versus differences in the fetal/adult environment, we used the Hardy scheme to sort fractions B-F B lineage cells from TdT-deficient BALB/c adult BM. V(H)7183-containing VDJCµ transcripts from these cells were amplified, cloned, sequenced, and compared with transcripts from wild-type perinatal liver and adult BM. The pattern of V(H)DJ(H) usage in TdT-deficient BM largely matched that of TdT-sufficient adult cells. What minor differences were detected in the pro-B cell stage tended to diminish with B cell maturation, suggesting strong environmental or Ag-driven pressure to achieve a specific range of V(H)DJ(H) usage regardless of the extent of N nucleotide addition. However, although the patterns of V(H)DJ(H) usage in the TdT-deficient B lineage cells paralleled that of wild-type adult cells, the length distribution, global amino acid composition, and charge distribution of the CDR-H3 repertoire proved to be a close, although not exact, homologue of the CDR-H3 repertoire first expressed by late pre-B cells in the TdT-insufficient perinatal liver. Thus, although differing in V(H) content, TdT-deficient mice appear to represent a good, although not perfect, model for testing the role of perinatal CDR-H3 limitations on late B cell development and Ab responses.