Hallmarks of improved immunological responses in the vaccination of more physically active elderly females.

Physical inactivity is one of the leading contributors to worldwide morbidity and mortality. The elderly are particularly susceptible since the features of physical inactivity overlap with the outcomes of natural aging - including the propensity to develop cardiovascular diseases, cancer, diabetes mellitus, sarcopenia and cognitive impairment. The age-dependent loss of immune function, or immunosenescence, refers to the progressive depletion of primary immune resources and is linked to the development of many of these conditions. Immunosenescence is primarily driven by chronic immune activation and physical activity interventions have demonstrated the potential to reduce the risk of complications in the elderly by modulating inflammation and augmenting the immune system. Since poor vaccination outcome is a hallmark of immunosenescence, the assessment of vaccine efficacy provides a window to study the immunological effects of regular physical activity. Using an accelerator-based study, we demonstrate in a Singaporean Chinese cohort that elderly women (n=56) who walk more after vaccination display greater post-vaccination expansion of monocytes and plasmablasts in peripheral blood. Active elderly female participants also demonstrated lower baseline levels of IP-10 and Eotaxin, and the upregulation of genes associated with monocyte/macrophage phagocytosis. We further describe postive correlations between the monocyte response and the post-vaccination H1N1 HAI titres of participants. Finally, active elderly women reveal a higher induction of antibodies against Flu B in their 18-month second vaccination follow-up. Altogether, our data are consistent with better immunological outcomes in those who are more physically active and highlight the pertinent contribution of monocyte activity.

Healthy elderly Singaporeans show no age-related humoral hyporesponsiveness nor diminished plasmablast generation in response to influenza vaccine.

ABSTRACT: Improving influenza vaccine efficacy is a priority to reduce the burden of influenza-associated morbidity and mortality. By careful selection of individuals based on health we show sustained response to influenza vaccination in older adults. Sustaining health in aging could be an important player in maintaining immune responses to influenza vaccination. TRIAL REGISTRATION: NCT03266237. Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT03266237.

Safety and Immunogenicity of Adenovirus 35 Tuberculosis Vaccine Candidate in Adults with Active or Previous Tuberculosis. A Randomized Trial.

RATIONALE: Administration of tuberculosis (TB) vaccines in participants with previous or current pulmonary TB may have the potential for causing harmful postvaccination immunologic (Koch-type) reactions. OBJECTIVES: To assess the safety and immunogenicity of three dose levels of the AERAS-402 live, replication-deficient adenovirus 35-vectored TB candidate vaccine, containing three mycobacterial antigens, in individuals with current or previous pulmonary TB. METHODS: We performed a phase II randomized, placebo-controlled, double-blinded dose-escalation study in an HIV-negative adult South African cohort (n = 72) with active pulmonary TB (on treatment for 1-4 mo) or pulmonary TB treated at least 12 months before study entry and considered cured. Safety endpoints included clinical assessment, flow volume curves, diffusing capacity of the lung for carbon monoxide, pulse oximetry, chest radiograph, and high-resolution thoracic computerized tomography scans. Cytokine expression by CD4 and CD8 T cells, after stimulation with Ag85A, Ag85B, and TB10.4 peptide pools, was examined by intracellular cytokine staining. MEASUREMENTS AND MAIN RESULTS: No apparent temporal or dose-related changes in clinical status (specifically acute, Koch phenomenon-like reactions), lung function, or radiology attributable to vaccine were observed. Injection site reactions were mild or moderate. Hematuria (by dipstick only) occurred in 25 (41%) of 61 AERAS-402 recipients and 3 (27%) of 11 placebo recipients, although no gross hematuria was reported. AERAS-402 induced robust CD8+ and moderate CD4+ T-cell responses, mainly to Ag85B in both vaccine groups. CONCLUSIONS: Administration of the AERAS-402 candidate TB vaccine to participants with current or previous pulmonary TB induced a robust immune response and is not associated with clinically significant pulmonary complications. Clinical trial registered with www.clinicaltrials.gov (NCT 02414828) and in the South African National Clinical Trials Register ( www.sanctr.gov.za DOH 27-0808-2060).

Human newborn bacille Calmette-Guérin vaccination and risk of tuberculosis disease: a case-control study.

BACKGROUND: An incomplete understanding of the immunological mechanisms underlying protection against tuberculosis (TB) hampers the development of new vaccines against TB. We aimed to define host correlates of prospective risk of TB disease following bacille Calmette-Guérin (BCG) vaccination. METHODS: In this study, 5,726 infants vaccinated with BCG at birth were enrolled. Host responses in blood collected at 10 weeks of age were compared between infants who developed pulmonary TB disease during 2 years of follow-up (cases) and those who remained healthy (controls). RESULTS: Comprehensive gene expression and cellular and soluble marker analysis failed to identify a correlate of risk. We showed that distinct host responses after BCG vaccination may be the reason: two major clusters of gene expression, with different myeloid and lymphoid activation and inflammatory patterns, were evident when all infants were examined together. Cases from each cluster demonstrated distinct patterns of gene expression, which were confirmed by cellular assays. CONCLUSIONS: Distinct patterns of host responses to Mycobacterium bovis BCG suggest that novel TB vaccines may also elicit distinct patterns of host responses. This diversity should be considered in future TB vaccine development.

Limited role for lymphotoxin α in the host immune response to Mycobacterium tuberculosis.

The contribution of lymphotoxin (LT)α in the host immune response to virulent Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin infections was investigated. Despite their ability to induce Th1 cytokine, IFN-γ, and IL-12 pulmonary response, "conventional" LTα(-/-) mice succumb rapidly to virulent M. tuberculosis aerosol infection, with uncontrolled bacilli growth, defective granuloma formation, necrosis, and reduced pulmonary inducible NO synthase expression, similar to TNF(-/-) mice. Contributions from developmental lymphoid abnormalities in LTα(-/-) mice were excluded because hematopoietic reconstitution with conventional LTα(-/-) bone marrow conferred enhanced susceptibility to wild-type mice, comparable to conventional LTα(-/-) control mice. However, conventional LTα(-/-) mice produced reduced levels of TNF after M. bovis bacillus Calmette-Guérin infection, and their lack of control of mycobacterial infection could be due to a defective contribution of either LTα or TNF, or both, to the host immune response. To address this point, the response of "neo-free" LTα(-/-) mice with unperturbed intrinsic TNF expression to M. tuberculosis infection was investigated in a direct comparative study with conventional LTα(-/-) mice. Strikingly, although conventional LTα(-/-) mice were highly sensitive, similar to TNF(-/-) mice, neo-free LTα(-/-) mice controlled acute M. tuberculosis infection essentially as wild-type mice. Pulmonary bacterial burden and inflammation was, however, slightly increased in neo-free LTα(-/-) mice 4-5 mo postinfection, but importantly, they did not succumb to infection. Our findings revise the notion that LTα might have a critical role in host defense to acute mycobacterial infection, independent of TNF, but suggest a contribution of LTα in the control of chronic M. tuberculosis infection.

Minimal Crossover between Mutations Associated with Omicron Variant of SARS-CoV-2 and CD8+ T-Cell Epitopes Identified in COVID-19 Convalescent Individuals.

There is a growing concern that ongoing evolution of SARS-CoV-2 could lead to variants of concern (VOC) that are capable of avoiding some or all of the multifaceted immune response generated by both prior infection or vaccination, with the recently described B.1.1.529 (Omicron) VOC being of particular interest. Peripheral blood mononuclear cell samples from PCR-confirmed, recovered COVID-19 convalescent individuals (n = 30) infected with SARS-CoV-2 in the United States collected in April and May 2020 who possessed at least one or more of six different HLA haplotypes were selected for examination of their anti-SARS-CoV-2 CD8+ T-cell responses using a multiplexed peptide-major histocompatibility complex tetramer staining approach. This analysis examined if the previously identified viral epitopes targeted by CD8+ T cells in these individuals (n = 52 distinct epitopes) are mutated in the newly described Omicron VOC (n = 50 mutations). Within this population, only one low-prevalence epitope from the Spike protein, restricted to two HLA alleles and found in 2/30 (7%) individuals, contained a single amino acid change associated with the Omicron VOC. These data suggest that virtually all individuals with existing anti-SARS-CoV-2 CD8+ T-cell responses should recognize the Omicron VOC and that SARS-CoV-2 has not evolved extensive T-cell escape mutations at this time. IMPORTANCE The newly identified Omicron variant of concern contains more mutations than any of the previous variants described to date. In addition, many of the mutations associated with the Omicron variant are found in areas that are likely bound by neutralizing antibodies, suggesting that the first line of immunological defense against COVID-19 is compromised. However, both natural infection and vaccination develop T-cell-based responses in addition to antibodies. This study examined if the parts of the virus, or epitopes, targeted by the CD8+ T-cell response in 30 individuals who recovered from COVID-19 in 2020 were mutated in the Omicron variant. Only one of 52 epitopes identified in this population contained an amino acid that was mutated in Omicron. These data suggest that the T-cell immune response in previously infected, and most likely vaccinated, individuals should still be effective against Omicron.

Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection.

The complement cascade defines an important link between the innate and the specific immune system. Here we show that mice deficient for the third component of complement (C3-/- mice) are highly susceptible to primary infection with influenza virus. C3-/- mice showed delayed viral clearance and increased viral titers in lung, whereas mice deficient for complement receptors CR1 and CR2 (Cr2-/- mice) cleared the infection normally. Priming of T-helper cells and cytotoxic T cells (CTLs) in lung-draining lymph nodes was reduced, and the recruitment into the lung of virus-specific CD4+ and CD8+ effector T cells producing interferon-gamma was severely impaired in C3-/- but not in Cr2-/- mice. Consequently, T-helper cell-dependent IgG responses were reduced in C3-/- mice but remained intact in Cr2-/- mice. These results demonstrate that complement induces specific immunity by promoting T-cell responses.

Specific T cell frequency and cytokine expression profile do not correlate with protection against tuberculosis after bacillus Calmette-Guérin vaccination of newborns.

RATIONALE: Immunogenicity of new tuberculosis (TB) vaccines is commonly assessed by measuring the frequency and cytokine expression profile of T cells. OBJECTIVES: We tested whether this outcome correlates with protection against childhood TB disease after newborn vaccination with bacillus Calmette-Guérin (BCG). METHODS: Whole blood from 10-week-old infants, routinely vaccinated with BCG at birth, was incubated with BCG for 12 hours, followed by cryopreservation for intracellular cytokine analysis. Infants were followed for 2 years to identify those who developed culture-positive TB-these infants were regarded as not protected against TB. Infants who did not develop TB disease despite exposure to TB in the household, and another group of randomly selected infants who were never evaluated for TB, were also identified-these groups were regarded as protected against TB. Cells from these groups were thawed, and CD4, CD8, and γδ T cell-specific expression of IFN-γ, TNF-α, IL-2, and IL-17 measured by flow cytometry. MEASUREMENTS AND MAIN RESULTS: A total of 5,662 infants were enrolled; 29 unprotected and two groups of 55 protected infants were identified. There was no difference in frequencies of BCG-specific CD4, CD8, and γδ T cells between the three groups of infants. Although BCG induced complex patterns of intracellular cytokine expression, there were no differences between protected and unprotected infants. CONCLUSIONS: The frequency and cytokine profile of mycobacteria-specific T cells did not correlate with protection against TB. Critical components of immunity against Mycobacterium tuberculosis, such as CD4 T cell IFN-γ production, may not necessarily translate into immune correlates of protection against TB disease.

The novel tuberculosis vaccine, AERAS-402, induces robust and polyfunctional CD4+ and CD8+ T cells in adults.

RATIONALE: AERAS-402 is a novel tuberculosis vaccine designed to boost immunity primed by bacillus Calmette-Guérin (BCG), the only licensed vaccine. OBJECTIVES: We investigated the safety and immunogenicity of AERAS-402 in healthy Mycobacterium tuberculosis-uninfected BCG-vaccinated adults from a tuberculosis-endemic region of South Africa. METHODS: Escalating doses of AERAS-402 vaccine were administered intramuscularly to each of three groups of healthy South African BCG-vaccinated adults, and a fourth group received two injections of the maximal dose. Participants were monitored for 6 months, with all adverse effects documented. Vaccine-induced CD4(+) and CD8(+) T-cell immunity was characterized by an intracellular cytokine staining assay of whole blood and peripheral blood mononuclear cells. MEASUREMENTS AND MAIN RESULTS: AERAS-402 was well tolerated, and no vaccine-related serious adverse events were recorded. The vaccine induced a robust CD4(+) T-cell response dominated by cells coexpressing IFN-gamma, tumor necrosis factor-alpha, and IL-2 ("polyfunctional" cells). AERAS-402 also induced a potent CD8(+) T-cell response, characterized by cells expressing IFN-gamma and/or tumor necrosis factor-alpha, which persisted for the duration of the study. CONCLUSIONS: Vaccination with AERAS-402 is safe and immunogenic in healthy adults. The immunity induced by the vaccine appears promising: polyfunctional T cells are thought to be important for protection against intracellular pathogens such as Mycobacterium tuberculosis, and evidence is accumulating that CD8(+) T cells are also important. AERAS-402 induced a robust and durable CD8(+) T-cell response, which appears extremely promising. Clinical trial registered with www.sanctr.gov.za (NHREC no. 1381).

CD8+ T-Cell Responses in COVID-19 Convalescent Individuals Target Conserved Epitopes From Multiple Prominent SARS-CoV-2 Circulating Variants.

This study examined whether CD8+ T-cell responses from coronavirus disease 2019 convalescent individuals (n = 30) potentially maintain recognition of the major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants (alpha, beta, gamma; n = 45 mutations assessed). Only 1 mutation found in Beta variant-spike overlapped with a previously identified epitope (1/52), suggesting that virtually all anti-SARS-CoV-2 CD8+ T-cell responses should recognize these newly described variants.

Minimal cross-over between mutations associated with Omicron variant of SARS-CoV-2 and CD8+ T cell epitopes identified in COVID-19 convalescent individuals.

There is a growing concern that ongoing evolution of SARS-CoV-2 could lead to variants of concern (VOC) that are capable of avoiding some or all of the multi-faceted immune response generated by both prior infection or vaccination, with the recently described B.1.1.529 (Omicron) VOC being of particular interest. Peripheral blood mononuclear cell samples from PCR-confirmed, recovered COVID-19 convalescent patients (n=30) infected with SARS-CoV-2 in the United States collected in April and May 2020 who possessed at least one or more of six different HLA haplotypes were selected for examination of their anti-SARS-CoV-2 CD8+ T-cell responses using a multiplexed peptide-MHC tetramer staining approach. This analysis examined if the previously identified viral epitopes targeted by CD8+ T-cells in these individuals (n=52 distinct epitopes) are mutated in the newly described Omicron VOC (n=50 mutations). Within this population, only one low-prevalence epitope from the Spike protein restricted to two HLA alleles and found in 2/30 (7%) individuals contained a single amino acid change associated with the Omicron VOC. These data suggest that virtually all individuals with existing anti-SARS-CoV-2 CD8+ T-cell responses should recognize the Omicron VOC, and that SARS-CoV-2 has not evolved extensive T-cell escape mutations at this time. IMPORTANCE: The newly identified Omicron variant of concern contains more mutations than any of the previous variants described to date. In addition, many of the mutations associated with the Omicron variant are found in areas that are likely bound by neutralizing antibodies, suggesting that the first line of immunological defense against COVID-19 may be compromised. However, both natural infection and vaccination develop T-cell based responses, in addition to antibodies. This study examined if the parts of the virus, or epitopes, targeted by the CD8+ T-cell response in thirty individuals who recovered from COVID-19 in 2020 were mutated in the Omicron variant. Only one of 52 epitopes identified in this population contained an amino acid that was mutated in Omicron. These data suggest that the T-cell immune response in previously infected, and most likely vaccinated individuals, should still be effective against Omicron.

CD8+ T cell responses in COVID-19 convalescent individuals target conserved epitopes from multiple prominent SARS-CoV-2 circulating variants.

This study examined whether CD8+ T-cell responses from COVID-19 convalescent individuals(n=30) potentially maintain recognition of the major SARS-CoV-2 variants. Out of 45 mutations assessed, only one from the B.1.351 Spike overlapped with a low-prevalence CD8+ epitope, suggesting that virtually all anti-SARS-CoV-2 CD8+ T-cell responses should recognize these newly described variants.

A Novel Sample Selection Approach to Aid the Identification of Factors That Correlate With the Control of HIV-1 Infection.

Individuals infected with HIV display varying rates of viral control and disease progression, with a small percentage of individuals being able to spontaneously control infection in the absence of treatment. In attempting to define the correlates associated with natural protection against HIV, extreme heterogeneity in the datasets generated from systems methodologies can be further complicated by the inherent variability encountered at the population, individual, cellular and molecular levels. Furthermore, such studies have been limited by the paucity of well-characterised samples and linked epidemiological data, including duration of infection and clinical outcomes. To address this, we selected 10 volunteers who rapidly and persistently controlled HIV, and 10 volunteers each, from two control groups who failed to control (based on set point viral loads) from an acute and early HIV prospective cohort from East and Southern Africa. A propensity score matching approach was applied to control for the influence of five factors (age, risk group, virus subtype, gender, and country) known to influence disease progression on causal observations. Fifty-two plasma proteins were assessed at two timepoints in the 1st year of infection. We independently confirmed factors known to influence disease progression such as the B*57 HLA Class I allele, and infecting virus Subtype. We demonstrated associations between circulating levels of MIP-1α and IL-17C, and the ability to control infection. IL-17C has not been described previously within the context of HIV control, making it an interesting target for future studies to understand HIV infection and transmission. An in-depth systems analysis is now underway to fully characterise host, viral and immunological factors contributing to control.

SARS-CoV-2-specific CD8+ T cell responses in convalescent COVID-19 individuals.

Characterization of the T cell response in individuals who recover from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 coronavirus disease 2019 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis and from humoral and inflammatory responses. There were 132 SARS-CoV-2-specific CD8+ T cell responses detected across 6 different HLAs, corresponding to 52 unique epitope reactivities. CD8+ T cell responses were detected in almost all convalescent individuals and were directed against several structural and nonstructural target epitopes from the entire SARS-CoV-2 proteome. A unique phenotype for SARS-CoV-2-specific T cells was observed that was distinct from other common virus-specific T cells detected in the same cross-sectional sample and characterized by early differentiation kinetics. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem cell and transitional memory states (subsets), which may be key to developing durable protection.

Systemic and Metabolic Signature of Sarcopenia in Community-Dwelling Older Adults.

BACKGROUND: Evidence suggests the pivotal contribution of nutrition as a modifiable risk factor for sarcopenia. The present cross-sectional study characterized the nutritional and metabolic profile of sarcopenia through an extensive exploration of a wide array of blood biomarkers related to muscle protein metabolism and transcriptomic signatures in community-dwelling elderly adults. METHODS: Among 189 older individuals with a mean age of 73.2 years, sarcopenia was diagnosed according to the Asian Working Group for Sarcopenia criteria based on appendicular lean mass measured by dual-energy X-ray absorptiometry scan, muscle strength, and gait speed. Nutritional status was evaluated using the mini-nutritional assessment (MNA). In addition, we assessed specific blood biomarkers of nutritional status (plasma essential amino acids [EAAs], vitamins), nicotine-derived metabolites, and an extensive microarray analysis from peripheral blood mononuclear cells. RESULTS: Malnutrition defined by low MNA score was independently associated with sarcopenia (p < .001). Sarcopenic elderly showed lower body mass index and leptin and higher adiponectin and high-density lipoproteins. Levels of EAAs including lysine, methionine, phenylalanine, threonine, as well as branched-chain AAs and choline, were inversely associated with sarcopenia. Furthermore, nicotine metabolites (cotinine and trans-3'-hydroxycotine) and vitamin B6 status were linked to one or more clinical and functional measures of sarcopenia. Differentially expressed genes and ingenuity pathway analysis supported the association of nutrition with sarcopenia. CONCLUSIONS: Herein, the characterization of a nutritional and metabolic signature of sarcopenia provides a firm basis and potential identification of specific targets and directions for the nutritional approach to the prevention and treatment of sarcopenia in aging populations.

CD8+ T cell responses in convalescent COVID-19 individuals target epitopes from the entire SARS-CoV-2 proteome and show kinetics of early differentiation.

Characterization of the T cell response in individuals who recover from SARS-CoV-2 infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 COVID-19 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis, humoral and inflammatory responses. 132 distinct SARS-CoV-2-specific CD8+ T cell epitope responses across six different HLAs were detected, corresponding to 52 unique reactivities. T cell responses were directed against several structural and non-structural virus proteins. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem-cell and transitional memory states, subsets, which may be key to developing durable protection.

Significantly skewed memory CD8+ T cell subsets in HIV-1 infected infants during the first year of life.

HIV-1 infection causes a severe T cell compromise; however, little is known about changes in naive, memory, effector and senescent T cell subsets during the first year of life. T cell subsets were studied over the first year of life in blood from 3 infant cohorts: untreated HIV-infected, HIV-exposed but uninfected, and HIV-unexposed. In HIV-infected infants, the frequency of CCR7(+)CD45RA(+) naive CD8(+) T cells was significantly decreased, while the frequency of CCR7(-)CD45RA(-) effector memory CD8(+) T cells was increased, compared with the control cohorts. A larger population of CD8(+) T cells in HIV-infected infants displayed a phenotype consistent with senescence. Differences in CD4(+) T cell subset frequencies were less pronounced, and no significant differences were observed between exposed and unexposed HIV-uninfected infants. We concluded that the proportion of naive, memory, effector and senescent CD8(+) T cells during the first year of life is significantly altered by HIV-1 infection.

Influenza Vaccine-Induced Antibody Responses Are Not Impaired by Frailty in the Community-Dwelling Elderly With Natural Influenza Exposure.

Background: Elderly adults over 65 years of age are recommended to receive seasonal influenza vaccination as they are at a higher risk of infection and its complications than the younger community. The elderly are often stratified according to frailty status where frail individuals are more susceptible to adverse health outcomes than their non-frail counterparts, however, it is not known whether immunity induced by influenza vaccination is impaired in the frail elderly. Study Design: Two hundred and five elderly subjects of Chinese ethnicity in Singapore (mean age 73.3 ± 5.3 years, 128 females and 77 males) were administered the recommended trivalent inactivated 2013-14 seasonal influenza vaccine (Vaxigrip™) containing A/H1N1, A/H3N2, and B strains. The elderly subjects were stratified into three groups according to Fried's frailty criteria (59 frail, 85 pre-frail, 61 robust) and were also ranked by Rockwood's frailty index (RFI). Statistical associations were evaluated between frailty status and pre- and post-vaccination antibody titres in sera measured by Hemagglutination inhibition (HAI) and microneutralization (MN) assays. Immunological responses across frailty strata were also studied in terms of leukocyte cellular distribution, cytokine levels and gene expression. Results: Post-vaccination, 83.4% of the subjects seroconverted for A/H1N1, 80.5% for A/H3N2, and 81% for the B strain. The seroconversion rates were comparable across frailty groups (A/H1N1, ANOVA, p = 0.7910; A/H3N2, ANOVA, p = 0.8356, B, ANOVA, p = 0.9741). Geometric mean titres of HAI and MN as well as seroprotection rates were also similar in all three frailty groups and uncorrelated with RFI (Spearman, r = 0.023, p = 0.738). No statistically significant differences were observed between the frailty groups in vaccine-induced modulation of leukocyte populations, cytokine responses, and gene expression profiles of peripheral blood mononuclear cells (PBMCs). Whereas, post- and pre-vaccination HAI titres were positively correlated after adjusting for age and gender (A/H1N1, R2 = 0.216, p = 9.1e-11; A/H3N2, R2 = 0.166, p = 3.4e-8; B, R2 = 0.104, p = 3.1e-5). With most subjects lacking previous history of influenza vaccination, the pre-vaccination titres were likely due to natural exposure and seen to match the pattern of influenza subtype prevalence in the time period of vaccination. Conclusion: The majority of the elderly subjects seroconverted for seasonal influenza upon vaccination, and importantly, influenza vaccination-induced humoral immune responses and seroprotection were similar across the frailty strata, indicating that frail individuals may also benefit from influenza vaccination. Pre-existing antibodies due to natural exposure appeared to positively influence vaccine-induced antibody responses.

Qualification of a whole blood intracellular cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity by flow cytometry.

BACKGROUND: Qualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity. METHODS: Whole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period. RESULTS: The limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-γ- and IL-2-expressing T cells and less than 0.01% for TNF-α- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4 °C or 20-23 °C) and time (45, 60 or 90 min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells. CONCLUSIONS: The whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.

Soluble TNFRp75 regulates host protective immunity against Mycobacterium tuberculosis.

Development of host protective immunity against Mycobacterium tuberculosis infection is critically dependent on the inflammatory cytokine TNF. TNF signals through 2 receptors, TNFRp55 and TNFRp75; however, the role of TNFRp75-dependent signaling in immune regulation is poorly defined. Here we found that mice lacking TNFRp75 exhibit greater control of M. tuberculosis infection compared with WT mice. TNFRp75-/- mice developed effective bactericidal granulomas and demonstrated increased pulmonary recruitment of activated DCs. Moreover, IL-12p40-dependent migration of DCs to lung draining LNs of infected TNFRp75-/- mice was substantially higher than that observed in WT M. tuberculosis-infected animals and was associated with enhanced frequencies of activated M. tuberculosis-specific IFN-γ-expressing CD4+ T cells. In WT mice, TNFRp75 shedding correlated with markedly reduced bioactive TNF levels and IL-12p40 expression. Neutralization of TNFRp75 in M. tuberculosis-infected WT BM-derived DCs (BMDCs) increased production of bioactive TNF and IL-12p40 to a level equivalent to that produced by TNFRp75-/- BMDCs. Addition of exogenous TNFRp75 to TNFRp75-/- BMDCs infected with M. tuberculosis decreased IL-12p40 synthesis, demonstrating that TNFRp75 shedding regulates DC activation. These data indicate that TNFRp75 shedding downmodulates protective immune function and reduces host resistance and survival; therefore, targeting TNFRp75 may be beneficial for improving disease outcome.