B cells discriminate HIV-1 Envelope protein affinities by sensing antigen binding association rates

HIV-1 Envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant, KD) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD, and whether B cells discriminate between proteins of similar affinities but that bind with different kinetic rates. Here we used a panel of Env proteins and Ramos B cell lines expressing IgM BCRs with specificity for CD4 binding-site broadly neutralizing (bnAb) or a precursor antibody to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD, but on sensing of association rate and a threshold antigen-BCR half-life.

SARS-CoV-2 specific B cell memory drives improved class switching and tissue homing responses to single dose Ad26.COV2.S vaccination in previously infected recipients

Neutralizing antibodies strongly correlate with protection for COVID-19 vaccines, but the corresponding memory B cells that form to protect against future infection are relatively understudied. Here we examine the effect of prior SARS-CoV-2 infection on the magnitude and phenotype of the B cell response to single dose Johnson and Johnson (Ad26.COV2.S) vaccination in South African health care workers. SARS-CoV-2 specific memory responses expand in response to Ad26.COV2.S and are maintained for the study duration (84 days) in all individuals. However, prior infection is associated with a greater frequency of these cells, a more prominent germinal center (GC) response, and increased class switched memory (CSM). These B cell features correlated with both neutralization and antibody-dependent cytotoxicity (ADCC) activity, and with the frequency of SARS-CoV-2 specific circulating T follicular helper cells (cTfh). In addition, the SARS-CoV-2 specific CD8+ T cell response correlated with increased memory B cell lung-homing, which was sustained in the infected group. Finally, although vaccination achieved equivalent B cell activation regardless of infection history, it was negatively impacted by age. These data show that phenotyping the B cell response to vaccination can provide mechanistic insight into the impact of prior infection on GC homing, CSM, cTfh, and neutralization activity. These data can provide early signals and mechanistic understanding to inform studies of vaccine boosting, durability, and co-morbidities.

A bacterium-like particle vaccine displaying Zika virus prM-E induces systemic immune responses in mice

The emergence of Zika virus (ZIKV) infection, which is unexpectedly associated with congenital defects, has prompted the development of safe and effective vaccines. The gram-positive enhancer matrix-protein anchor (GEM-PA) display system has emerged as a versatile and highly effective platform for delivering target proteins for vaccines. In this article, we developed a bacterium-like particle vaccine ZI-△-PA-GEM based on the GEM-PA system. The fusion protein ZI-△-PA, which contains the prM-E-△ protein of ZIKV (with a stem-transmembrane region deletion) and the protein anchor PA3, was expressed. The fusion protein was successfully displayed on the GEM surface, forming ZI-△-PA-GEM. Moreover, when BALB/c mice were immunized intramuscularly with ZI-△-PA-GEM combined with 201 VG and poly(I:C) adjuvants, durable ZIKV-specific IgG and protective neutralizing antibody responses were induced. Potent B cell/DC activation was also be stimulated early after immunization. Remarkably, splenocyte proliferation, the secretion of multiple cytokines, T/B cell activation and central memory T cell responses were elicited. These data indicate that ZI-△-PA-GEM is a promising bacterium-like particle vaccine candidate for ZIKV.

M-CSF-Stimulated CD11b+ Myeloid Cells Induce Alopecia Areata in C3H/HeJ Mice

Alopecia areata (AA) is a T cell-mediated autoimmune skin disease with clinical features of hair loss and skin inflammation. It is unclear whether other immune cells except T lymphocytes are also involved in the development of AA. Here, our results reveal that dermal injection of either CD11b+ myeloid cells isolated from AA-affected skin or non-AA splenocyte-derived CD11b+ cells treated with macrophage colony-stimulating factor (M-CSF) induces AA in C3H/HeJ mice. The functional similarity of these cells in induction of AA seems to be due to a higher expression of M-CSF in AA affected skin. To explore the mechanism by which dermal injection of CD11b+ cells induce AA, we co-culture either AA derived skin cells or M-CSF-stimulated CD11b+ cells with naïve splenocytes. The results of splenocyte proliferation assay and immunoglobulin release in conditioned medium show a significant increase of splenocyte proliferation and IgG level in conditioned medium under both conditions as compared to controls. Most activated splenocytes induced by M-CSF-stimulated myeloid cells are B lymphocytes. B cell activation are further confirmed in AA-affected skin and skin draining lymph nodes of AA mice. In conclusion, in this study, we have provided evidence that M-CSF stimulated CD11b+ cells are able to induce AA in C3H/HeJ mice through a possible mechanism by activating B lymphocytes. This finding may provide insight for understanding the pathogenesis of AA.

The EBNA2-EBF1 complex promotes oncogenic MYC expression levels and metabolic processes required for cell cycle progression of Epstein-Barr virus-infected B cells

Epstein-Barr virus (EBV) is a human tumor virus, which preferentially infects resting human B cells. Upon infection in vitro, EBV activates and immortalizes these cells. The viral latent protein EBV nuclear antigen (EBNA) 2 is essential for B cell activation and immortalization; it targets and binds the cellular and ubiquitously expressed DNA binding protein CBF1, thereby transactivating a plethora of viral and cellular genes. In addition, EBNA2 uses its N-terminal dimerization (END) domain to bind early B cell factor (EBF) 1, a pioneer transcription factor specifying the B cell lineage. We found that EBNA2 exploits EBF1 to support key metabolic processes and to foster cell cycle progression of infected B cells in their first cell cycles upon activation. An α1-helix within the END domain was found to promote EBF1 binding. EBV mutants lacking the α1-helix in EBNA2 can infect and activate B cells efficiently, but the activated cells fail to complete the early S phase of their initial cell cycle. Expression of MYC, target genes of MYC and E2F as well as multiple metabolic processes linked to cell cycle progression are impaired in EBV∆α1 infected B cells. Our findings indicate that EBF1 controls B cell activation via EBNA2 and, thus, has a critical role in regulating the cell cycle of EBV infected B cells. This is a function of EBF1 going beyond its well-known contribution to B cell lineage specification.

Safety of Multiple Vaccinations and Durability of Vaccine-Induced Antibodies in an Italian Military Cohort 5 Years after Immunization

We previously examined the safety and immunogenicity of multiple vaccines administered to a military cohort, divided into two groups, the first composed of students at military schools, thus operating inside the national borders for at least 3 years, and the other formed of soldiers periodically engaged in a 9-month-long mission abroad (Lebanon). In the current study, we analyzed 112 individuals of this cohort, 50 pertaining to the first group and 62 to the second group, in order to examine the possible late appearance of side effects and to calculate the half-life of the induced antibodies. Moreover, the possible involvement of B-cell polyclonal activation as a pathogenetic mechanism for long term antibody persistence has even been explored. No late side effects, as far as autoimmunity and/or lymphoproliferation appearance, have been noticed. The long duration of the vaccine induced anti-HAV antibodies has been confirmed, whereas the antibodies induced by tetravalent meningococcal polysaccharide vaccine have been found to persist above the threshold for putative protection for a longer time, and anti-tetanus, diphtheria, and polio 1 and 3 for a shorter time than previously estimated. No signs of polyclonal B-cell activation have been found, as a possible mechanism to understand the long antibody persistence.

Zeolitic Imidazolate Framework Nanoencapsulation of CpG for Stabilization and Enhancement of Immunoadjuvancy

Metal-organic frameworks (MOFs) have been used to improve vaccine formulations by stabilizing proteins and protecting them against thermal degradation. This has led to increased 2 immunogenicity of these proteinaceous therapeutics. In this work we show that MOFs can also be used to protect the ssDNA oligomer, CpG, to increase its immunoadjuvancy. By encapsulating phosphodiester CpG in the zinc-based MOF, ZIF-8, the DNA oligomer is protected from nuclease degradation and exhibits improved cellular uptake. As a result, we have been able to achieve drastically enhanced B-cell activation in splenocyte cultures comparable to the current state-of-the-art, phosphorothioate CpG. Furthermore, we have made a direct comparison of micro- and nano-sized MOF for the optimization of particulate delivery of immunoadjuvants to maximize immune activation.

Adjuvant Activity of CpG-Oligonucleotide Administered Transcutaneously in Combination with Vaccination Using a Self-Dissolving Microneedle Patch in Mice

In this study, we investigated the mechanism of transcutaneous adjuvant activity of the CpG-oligonucleotide (K3) in mice. Transcutaneous immunization (TCI) with an ovalbumin-loaded self-dissolving microneedle patch (OVA-sdMN) and K3-loaded hydrophilic gel patch (HG) increased OVA-specific Th2- and Th1-type IgG subclass antibody titers more rapidly and strongly than those after only OVA-sdMN administration. However, the antigen-specific proliferation of OVA-specific CD4+ T cells was similar between the OVA-only and the OVA+K3 groups. Population analysis of various immune cells in draining lymph nodes (dLNs) in the primary immune response revealed that the OVA+K3 combination doubled the number of dLN cells, with the most significant increase in B cells. Phenotypic analysis by flow cytometry revealed that B-cell activation and maturation were promoted in the OVA+K3 group, suggesting that direct B-cell activation by K3 largely contributed to the rapid increase in antigen-specific antibody titer in TCI. In the secondary immune response, a significant increase in effector T cells and effector memory T cells, and an increase in memory B cells were observed in the OVA+K3 group compared with that in the OVA-only group. Thus, K3, as a transcutaneous adjuvant, can promote the memory differentiation of T and B cells.