Integrated antibody and cellular immunity monitoring are required for assessment of the long term protection that will be essential for effective next generation vaccine development.

The COVID pandemic exposed the critical role T cells play in initial immunity, the establishment and maintenance of long term protection, and of durable responsiveness against novel viral variants. A growing body of evidence indicates that adding measures of cellular immunity will fill an important knowledge gap in vaccine clinical trials, likely leading to improvements in the effectiveness of the next generation vaccines against current and emerging variants. In depth cellular immune monitoring in Phase II trials, particularly for high risk populations such as the elderly or immune compromised, should result in better understanding of the dynamics and requirements for establishing effective long term protection. Such analyses can result in cellular immunity correlates that can then be deployed in Phase III studies using appropriate, scalable technologies. Measures of cellular immunity are less established than antibodies as correlates of clinical immunity, and some misconceptions persist about cellular immune monitoring usefulness, cost, complexity, feasibility, and scalability. We outline the currently available cellular immunity assays, review their readiness for use in clinical trials, their logistical requirements, and the type of information each assay generates. The objective is to provide a reliable source of information that could be leveraged to develop a rational approach for comprehensive immune monitoring during vaccine development.

SARS-CoV-2 specific immune responses in overweight and obese COVID-19 patients.

Obesity is a known risk factor for severe respiratory tract infections. In this prospective study, we assessed the impact of being obese or overweight on longitudinal SARS-CoV-2 humoral and cellular responses up to 18 months after infection. 274 patients provided blood samples at regular time intervals up to 18 months including obese (BMI ≥30, n=32), overweight (BMI 25-29.9, n=103) and normal body weight (BMI 18.5-24.9, n=134) SARS-CoV-2 patients. We determined SARS-CoV-2 spike-specific IgG, IgA, IgM levels by ELISA and neutralising antibody titres by neutralisation assay. RBD- and spike-specific memory B cells were investigated by ELISpot, spike- and non-spike-specific IFN-γ, IL-2 and IFN-γ/IL-2 secreting T cells by FluoroSpot and T cell receptor (TCR) sequencing was performed. Higher BMI correlated with increased COVID-19 severity. Humoral and cellular responses were stronger in overweight and obese patients than normal weight patients and associated with higher spike-specific IgG binding titres relative to neutralising antibody titres. Linear regression models demonstrated that BMI, age and COVID-19 severity correlated independently with higher SARS-CoV-2 immune responses. We found an increased proportion of unique SARS-CoV-2 specific T cell clonotypes after infection in overweight and obese patients. COVID-19 vaccination boosted humoral and cellular responses irrespective of BMI, although stronger immune boosting was observed in normal weight patients. Overall, our results highlight more severe disease and an over-reactivity of the immune system in overweight and obese patients after SARS-CoV-2 infection, underscoring the importance of recognizing overweight/obese individuals as a risk group for prioritisation for COVID-19 vaccination.

Durable immune responses after BNT162b2 vaccination in home-dwelling old adults.

Objectives: Elderly are an understudied, high-risk group vulnerable to severe COVID-19. We comprehensively analyzed the durability of humoral and cellular immune responses after BNT162b2 vaccination and SARS-CoV-2 infection in elderly and younger adults. Methods: Home-dwelling old (n = 100, median 86 years) and younger adults (n = 449, median 38 years) were vaccinated with two doses of BNT162b2 vaccine at 3-week intervals and followed for 9-months. Vaccine-induced responses were compared to home-isolated COVID-19 patients (n = 183, median 47 years). Our analysis included neutralizing antibodies, spike-specific IgG, memory B-cells, IFN-γ and IL-2 secreting T-cells and sequencing of the T-cell receptor (TCR) repertoire. Results: Spike-specific breadth and depth of the CD4+ and CD8+ TCR repertoires were significantly lower in the elderly after one and two vaccinations. Both vaccinations boosted IFN-γ and IL-2 secreting spike-specific T-cells responses, with 96 % of the elderly and 100 % of the younger adults responding after the second dose, although responses were not maintained at 9-months. In contrast, T-cell responses persisted up to 12-months in infected patients. Spike-specific memory B-cells were induced after the first dose in 87 % of the younger adults compared to 38 % of the elderly, which increased to 83 % after the second dose. Memory B-cells were maintained at 9-months post-vaccination in both vaccination groups. Neutralizing antibody titers were estimated to last for 1-year in younger adults but only 6-months in the older vaccinees. Interestingly, infected older patients (n = 15, median 75 years) had more durable neutralizing titers estimated to last 14-months, 8-months longer than the older vaccinees. Conclusions: Vaccine-induced spike-specific IgG and neutralizing antibodies were consistently lower in the older than younger vaccinees. Overall, our data provide valuable insights into the kinetics of the humoral and cellular immune response in the elderly after SARS-CoV-2 vaccination or infection, highlighting the need for two doses, which can guide future vaccine design.Clinical trials.gov; NCT04706390.

Characterisation of T cell receptor repertoires in coeliac disease.

AIMS: Characterise T-cell receptor gene (TR) repertoires of small intestinal T cells of patients with newly diagnosed (active) coeliac disease (ACD), refractory CD type I (RCD I) and patients with CD on a gluten-free diet (GFD). METHODS: Next-generation sequencing of complementarity-determining region 3 (CDR3) of rearranged T cell receptor ß (TRB) and γ (TRG) genes was performed using DNA extracted from intraepithelial cell (IEC) and lamina propria cell (LPC) fractions and a small subset of peripheral blood mononuclear cell (PBMC) samples obtained from CD and non-CD (control) patients. Several parameters were assessed, including relative abundance and enrichment. RESULTS: TRB and TRG repertoires of CD IEC and LPC samples demonstrated lower clonality but higher frequency of rearranged TRs compared with controls. No CD-related differences were detected in the limited number of PBMC samples. Previously published LP gliadin-specific TRB sequences were more frequently detected in LPC samples from patients with CD compared with non-CD controls. TRG repertoires of IECs from both ACD and GFD patients demonstrated increased abundance of certain CDR3 amino acid (AA) motifs compared with controls, which were encoded by multiple nucleotide variants, including one motif that was enriched in duodenal IECs versus the PBMCs of CD patients. CONCLUSIONS: Small intestinal TRB and TRG repertoires of patients with CD are more diverse than individuals without CD, likely due to mucosal recruitment and accumulation of T cells because of protracted inflammation. Enrichment of the unique TRG CDR3 AA sequence in the mucosa of patients with CD may suggest disease-associated changes in the TCRγδ IE lymphocyte (IEL) landscape.

Cutting Edge: Bacillus Calmette-Guérin-Induced T Cells Shape Mycobacterium tuberculosis Infection before Reducing the Bacterial Burden.

Growing evidence suggests the outcome of Mycobacterium tuberculosis infection is established rapidly after exposure, but how the current tuberculosis vaccine, bacillus Calmette-Guérin (BCG), impacts early immunity is poorly understood. In this study, we found that murine BCG immunization promotes a dramatic shift in infected cell types. Although alveolar macrophages are the major infected cell for the first 2 weeks in unimmunized animals, BCG promotes the accelerated recruitment and infection of lung-infiltrating phagocytes. Interestingly, this shift is dependent on CD4 T cells, yet does not require intrinsic recognition of Ag presented by infected alveolar macrophages. M. tuberculosis-specific T cells are first activated in lung regions devoid of infected cells, and these events precede vaccine-induced reduction of the bacterial burden, which occurs only after the colocalization of T cells and infected cells. Understanding how BCG alters early immune responses to M. tuberculosis provides new avenues to improve upon the immunity it confers.

Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis.

CD4 T cells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 T cells have conferred little or no protection. Here we examined CD4 T cell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific T cells to be more differentiated than Ag85B-specific T cells. The ability of each T cell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific T cells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific T cells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required to optimize protection mediated by T cells recognizing antigens expressed at distinct stages of Mtb infection.

MiR-155-regulated molecular network orchestrates cell fate in the innate and adaptive immune response to Mycobacterium tuberculosis.

The regulation of host-pathogen interactions during Mycobacterium tuberculosis (Mtb) infection remains unresolved. MicroRNAs (miRNAs) are important regulators of the immune system, and so we used a systems biology approach to construct an miRNA regulatory network activated in macrophages during Mtb infection. Our network comprises 77 putative miRNAs that are associated with temporal gene expression signatures in macrophages early after Mtb infection. In this study, we demonstrate a dual role for one of these regulators, miR-155. On the one hand, miR-155 maintains the survival of Mtb-infected macrophages, thereby providing a niche favoring bacterial replication; on the other hand, miR-155 promotes the survival and function of Mtb-specific T cells, enabling an effective adaptive immune response. MiR-155-induced cell survival is mediated through the SH2 domain-containing inositol 5-phosphatase 1 (SHIP1)/protein kinase B (Akt) pathway. Thus, dual regulation of the same cell survival pathway in innate and adaptive immune cells leads to vastly different outcomes with respect to bacterial containment.

Interdependence between Interleukin-1 and Tumor Necrosis Factor Regulates TNF-Dependent Control of Mycobacterium tuberculosis Infection.

The interleukin-1 receptor I (IL-1RI) is critical for host resistance to Mycobacterium tuberculosis (Mtb), yet the mechanisms of IL-1RI-mediated pathogen control remain unclear. Here, we show that without IL-1RI, Mtb-infected newly recruited Ly6G(hi) myeloid cells failed to upregulate tumor necrosis factor receptor I (TNF-RI) and to produce reactive oxygen species, resulting in compromised pathogen control. Furthermore, simultaneous ablation of IL-1RI and TNF-RI signaling on either stroma or hematopoietic cells led to early lethality, indicating non-redundant and synergistic roles of IL-1 and TNF in mediating macrophage-stroma cross-talk that was critical for optimal control of Mtb infection. Finally, we show that even in the presence of functional Mtb-specific adaptive immunity, the lack of IL-1α and not IL-1ß led to an exuberant intracellular pathogen replication and progressive non-resolving inflammation. Our study reveals functional interdependence between IL-1 and TNF in enabling Mtb control mechanisms that are critical for host survival.

ICOS and Bcl6-dependent pathways maintain a CD4 T cell population with memory-like properties during tuberculosis.

Immune control of persistent infection with Mycobacterium tuberculosis (Mtb) requires a sustained pathogen-specific CD4 T cell response; however, the molecular pathways governing the generation and maintenance of Mtb protective CD4 T cells are poorly understood. Using MHCII tetramers, we show that Mtb-specific CD4 T cells are subject to ongoing antigenic stimulation. Despite this chronic stimulation, a subset of PD-1(+) cells is maintained within the lung parenchyma during tuberculosis (TB). When transferred into uninfected animals, these cells persist, mount a robust recall response, and provide superior protection to Mtb rechallenge when compared to terminally differentiated Th1 cells that reside preferentially in the lung-associated vasculature. The PD-1(+) cells share features with memory CD4 T cells in that their generation and maintenance requires intrinsic Bcl6 and intrinsic ICOS expression. Thus, the molecular pathways required to maintain Mtb-specific CD4 T cells during ongoing infection are similar to those that maintain memory CD4 T cells in scenarios of antigen deprivation. These results suggest that vaccination strategies targeting the ICOS and Bcl6 pathways in CD4 T cells may provide new avenues to prevent TB.

Pathogen-specific Treg cells expand early during mycobacterium tuberculosis infection but are later eliminated in response to Interleukin-12.

Thymically derived Foxp3⁺ regulatory T (Treg) cells have a propensity to recognize self-peptide:MHC complexes, but their ability to respond to epitope-defined foreign antigens during infectious challenge has not been demonstrated. Here we show that pulmonary infection with Mycobacterium tuberculosis (Mtb), but not Listeria monocytogenes (Lm), induced robust lymph node expansion of a highly activated population of pathogen-specific Treg cells from the pre-existing pool of thymically derived Treg cells. These antigen-specific Treg cells peaked in numbers 3 weeks after infection but subsequently underwent selective elimination driven, in part, by interleukin-12-induced intrinsic expression of the Th1-cell-promoting transcription factor T-bet. Thus, the initial Mtb-induced inflammatory response promotes pathogen-specific Treg cell proliferation, but these cells are actively culled later, probably to prevent suppression during later stages of infection. These findings have important implications for the prevention and treatment of tuberculosis and other chronic diseases in which antigen-specific Treg cells restrict immunity.

Foxp3(+) regulatory T cells in tuberculosis.

The immune response to Mycobacterium tuberculosis (Mtb) must be tightly regulated to mount a sufficient response to limit bacterial growth and dissemination while avoiding excessive inflammation that could damage host tissues. A wide variety of cell types, cell surface molecules, and cytokines are likely to contribute to this regulation, but recent studies have revealed that a subset of CD4 T cells expressing the transcription factor Foxp3, called regulatory T (reg) cells, play a critical role [1-3]. Although the first reports of T reg cells in tuberculosis (TB) occurred only recently (i.e., 2006) [4, 5], we have already gained many insights into their activity during TB. While it is likely that T reg cells do play some beneficial roles by preventing inflammation-mediated damage to host tissues during TB, this aspect of their function has not been well studied to date. What is clear, however, is that during the initial T cell response to Mtb infection, Mtb induces the expansions of T reg cells that delay the onset of adaptive immunity, suggesting that Mtb has hijacked T reg cell-mediated immune suppression to allow it to replicate unabated in the lung until T cells finally arrive [6]. In this chapter, we will first provide an overview of the delayed T cell response to Mtb and a brief introduction to regulatory T cells. We will then review what is known about T reg cells from observations in human populations, discuss mechanistic insights revealed in the mouse model, and speculate about the relevance of this understanding for future efforts to prevent and treat TB.

Distinct functions of antigen-specific CD4 T cells during murine Mycobacterium tuberculosis infection.

The immune response elicited after Mycobacterium tuberculosis (Mtb) infection is critically dependent on CD4 T cells during both acute and chronic infection. How CD4 T-cell responses are maintained throughout infection is not well understood, and evidence from other infection models has suggested that, under conditions of chronic antigen stimulation, T cells can undergo replicative exhaustion. These findings led us to determine whether subpopulations of CD4 T cells existed that displayed markers of terminal differentiation or exhaustion during murine Mtb infection. Analysis of antigen-specific effector CD4 T cells revealed that programmed death-1 (PD-1) and the killer cell lectin-like receptor G1 (KLRG1) delineated subpopulations of T cells. PD-1-expressing CD4 T cells were highly proliferative, whereas KLRG1 cells exhibited a short lifespan and secreted the cytokines IFNγ and TNFα. Adoptive transfer studies demonstrated that proliferating PD-1-positive CD4 T cells differentiated into cytokine-secreting KLRG1-positive T cells, but not vice versa. Thus, proliferating PD-1-positive cells are not exhausted, but appear to be central to maintaining antigen-specific effector T cells during chronic Mtb infection. Our findings suggest that antigen-specific T-cell responses are maintained during chronic mycobacterial infection through the continual production of terminal effector cells from a proliferating precursor population.

Fidelity of pathogen-specific CD4+ T cells to the Th1 lineage is controlled by exogenous cytokines, interferon-gamma expression, and pathogen lifestyle.

The degree of lineage stability achieved by pathogen-specific CD4(+) T cells in vivo, and how this impacts host defense against infection, remains unclear. We demonstrate that in response to Th1-polarizing intracellular bacterial or viral pathogens, only 80%-90% of responding polyclonal T cells become indelibly committed to this lineage. Th1 commitment was nearly invariant in cells that proliferated extensively, but perturbations to the extrinsic cytokine milieu or the pathogen's ability to enter the cytosol impeded commitment and promoted plasticity for future IL-17 expression. Conversely, cell-intrinsic interferon-gamma expression and acquisition of permissive chromatin at the Ifng gene during priming predicted heritable Th1 commitment. Importantly, CD4(+) T cells that retained plasticity conferred protection against Mycobacterium tuberculosis, while these protective effects were abolished with Th17 polarization. These findings illustrate the immune signals that induce memory CD4(+) T cell responses required for maintaining host defense against infection yet are adaptable in novel environmental contexts.

Pathogen-specific regulatory T cells delay the arrival of effector T cells in the lung during early tuberculosis.

The ability of the adaptive immune system to restrict Mycobacterium tuberculosis (Mtb) is impeded by activated Foxp3(+) regulatory T (T reg) cells. The importance of pathogen-specific T reg cells in this process has not been addressed. We show that T reg cell expansion after aerosol Mtb infection does not occur until Mtb is transported to the pulmonary lymph node (pLN), and Mtb-specific T reg cells have an increased propensity to proliferate. Even small numbers of Mtb-specific T reg cells are capable of delaying the priming of effector CD4(+) and CD8(+) T cells in the pLN and their subsequent accumulation in the lung, the primary site of infection. This delay likely prolongs the initial phase of bacterial expansion and explains the higher bacterial burden observed in these mice. Thus, T reg cells recognizing Mtb-derived antigens specifically and potently restrict protective immune responses during tuberculosis.

Expansion and function of Foxp3-expressing T regulatory cells during tuberculosis.

Mycobacterium tuberculosis (Mtb) frequently establishes persistent infections that may be facilitated by mechanisms that dampen immunity. T regulatory (T reg) cells, a subset of CD4(+) T cells that are essential for preventing autoimmunity, can also suppress antimicrobial immune responses. We use Foxp3-GFP mice to track the activity of T reg cells after aerosol infection with Mtb. We report that during tuberculosis, T reg cells proliferate in the pulmonary lymph nodes (pLNs), change their cell surface phenotype, and accumulate in the pLNs and lung at a rate parallel to the accumulation of effector T cells. In the Mtb-infected lung, T reg cells accumulate in high numbers in all sites where CD4(+) T cells are found, including perivascular/peribronchiolar regions and within lymphoid aggregates of granulomas. To determine the role of T reg cells in the immune response to tuberculosis, we generated mixed bone marrow chimeric mice in which all cells capable of expressing Foxp3 expressed Thy1.1. When T reg cells were depleted by administration of anti-Thy1.1 before aerosol infection with Mtb, we observed approximately 1 log less of colony-forming units of Mtb in the lungs. Thus, after aerosol infection, T reg cells proliferate and accumulate at sites of infection, and have the capacity to suppress immune responses that contribute to the control of Mtb.

Epidemiology and molecular characterization of vancomycin resistant Enterococci isolates in India.

Little attention has been paid to the problem of the spread of vancomycin resistant enterococci (VRE) in India. Between August 2002 to March 2003, faecal and urine samples of patients from various wards of the Postgraduate Institute of Medical Education and Research, Chandigarh, India, were screened for vancomycin resistance. 36 VRE were isolated (18 Enterococcus gallinarum, 9 E. casseliflavus, 7 E. faecium and 2 E. faecalis). These isolates were characterized as low-, moderate- and high-level resistant strains by phenotypic as well as genotypic methods such as minimum inhibitory concentration determination, polymerase chain reaction assays, sequencing of PCR products and multiple sequence alignment of van genes. Correlations established between these results and the vancomycin resistance markers were designated as vanA (783 bp), vanB (635 bp), vanC1 (822 bp) and vanC2 (484 bp) according to the findings of earlier workers as well as comparison with existing databases. Prolonged hospital stay and vancomycin were important risk factors for both VRE UTI and colonization. Renal dialysis, renal failure, prior aminoglycoside and third generation cephalosporin were the other significant factors for VRE UTI. The study also highlights the importance of screening for VRE in clinical samples and recommends the institution of control measures to prevent the further spread of VRE.