Understanding the impact of age on vaccinations is essential for the design and delivery of vaccines against SARS-CoV-2. Here, we present findings from a comprehensive analysis of multiple compartments of the memory immune response in 312 individuals vaccinated with the BNT162b2 SARS-CoV-2 mRNA vaccine. Two vaccine doses induce high antibody and T cell responses in most individuals. However, antibody recognition of the Spike protein of the Delta and Omicron variants is less efficient than that of the ancestral Wuhan strain. Age-stratified analyses identify a group of low antibody responders where individuals ≥60 years are overrepresented. Waning of the antibody and cellular responses is observed in 30% of the vaccinees after 6 months. However, age does not influence the waning of these responses. Taken together, while individuals ≥60 years old take longer to acquire vaccine-induced immunity, they develop more sustained acquired immunity at 6 months post-vaccination. A third dose strongly boosts the low antibody responses in the older individuals against the ancestral Wuhan strain, Delta and Omicron variants.

The SARS‐CoV‐2 Delta (B.1.617.2) variant is capable of infecting vaccinated persons. An open question remains as to whether deficiencies in specific vaccine‐elicited immune responses result in susceptibility to vaccine breakthrough infection. We investigated 55 vaccine breakthrough infection cases (mostly Delta) in Singapore, comparing them against 86 vaccinated close contacts who did not contract infection. Vaccine breakthrough cases showed lower memory B cell frequencies against SARS‐CoV‐2 receptor‐binding domain (RBD). Compared to plasma antibodies, antibodies secreted by memory B cells retained a higher fraction of neutralizing properties against the Delta variant. Inflammatory cytokines including IL‐1β and TNF were lower in vaccine breakthrough infections than primary infection of similar disease severity, underscoring the usefulness of vaccination in preventing inflammation. This report highlights the importance of memory B cells against vaccine breakthrough and suggests that lower memory B cell levels may be a correlate of risk for Delta vaccine breakthrough infection. This study compares the immune characteristics of 55 patients with vaccine breakthrough SARS‐CoV‐2 infection and 86 uninfected vaccinated close contacts. Memory B cell levels differed between the groups, identifying them as potential immune correlates of protection in Delta vaccine breakthrough. This study compares the immune characteristics of 55 patients with vaccine breakthrough SARS‐CoV‐2 infection and 86 uninfected vaccinated close contacts. Memory B cell levels differed between the groups, identifying them as potential immune correlates of protection in Delta vaccine breakthrough.

Significance GTPases play essential roles in living organisms, functioning as molecular switches or timers in many fundamental cellular processes. HflX is a GTPase that is highly conserved in the entire prokaryotic kingdom and is involved in the stress response of bacteria. We show here that under hypoxic stress, by modulating the cell’s translational activity, HflX controls mycobacteria growth rate and entry into nonreplicating state. Our work assigns a physiological role to HflX in pathogenic mycobacteria, implicating this factor in one of the most elusive but critical phenomena of replication arrest, which is responsible for the worldwide TB drug resistance crisis and a major roadblock to successful shorter TB therapies.

Early detection of infection is crucial to limit the spread of coronavirus disease 2019 (COVID-19). Here we develop a flow cytometry-based assay to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein antibodies in individuals with COVID-19. The assay detects specific immunoglobulin M (IgM), IgA, and IgG in individuals with COVID-19 and also acquisition of all IgG subclasses, with IgG1 being the most dominant. The antibody response is significantly higher at a later stage of infection. Furthermore, asymptomatic individuals with COVID-19 also develop specific IgM, IgA, and IgG, with IgG1 being the most dominant subclass. Although the antibody levels are lower in asymptomatic infection, the assay is highly sensitive and detects 97% of asymptomatic infections. These findings demonstrate that the assay can be used for serological analysis of symptomatic and asymptomatic infections, which may otherwise remain undetected.

GTPase HflX is highly conserved in prokaryotes and is a ribosome splitting factor during heat shock in E. coli. Here we report that HflX produced by slow growing M. tuberculosis and M. bovis BCG is a GTPase that plays a critical role in the pathogen’s transition to a non-replicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient M. bovis BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model, that precipitated entry into dormancy. The KO displayed the hallmarks of dormant mycobacteria including phenotypic drug resistance, altered morphology, low intracellular ATP and up-regulated dormancy dos regulon. KO-infected mice displayed increased bacterial burden during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic phase. Unlike fast-growing mycobacteria, BCG HlfX was not involved in antibiotic resistance under normoxia. Proteomics, pull-down and ribo-sequencing supported that mycobacterial HflX is a ribosome binding protein that controls the translational activity of the cell. Collectively, our study provides further insights into the mechanisms deployed by mycobacteria to adapt to their hypoxic microenvironment.

Tuberculosis (TB) pathogenesis is characterized by inadequate immune cell activation and delayed T cell response in the host. Recent immunotherapeutic efforts have been directed at stimulating innate immunity and enhancing interactions between antigen presenting cells and T cells subsets to improve the protective immunity against TB. In this study, we investigated the immunostimulatory properties of bacterial ghosts (BG) as a novel approach to potentiate the host immunity against mycobacterial infection. BG are intact cytoplasm-free Escherichia coli envelopes and have been developed as bacterial vaccines and adjuvant/delivery system in cancer immunotherapy. However, BG have yet to be exploited as immunopotentiators in the context of infectious diseases. Here, we showed that BG are potent inducers of dendritic cells (DC), which led to enhanced T cell proliferation and differentiation into effector cells. BG also induced macrophage activation, which was associated with enhanced nitric oxide production, a key anti-mycobacterial weapon. We further demonstrated that the immunostimulatory capability of BG far exceeds that of LPS and involves both TLR4-dependent and independent pathways. Consistently, BG treatment, but not LPS treatment, reduced the bacterial burden in infected mice, which correlated with increased influx of innate and adaptive effector immune cells and increased production of key cytokines in the lungs. Finally and importantly, enhanced bacilli killing was seen in mice co-administered with BG and second-line TB drugs bedaquiline and delamanid. Overall, this work paves the way for BG as potent immunostimulators that may be harnessed to improve mycobacteria killing at the site of infection.