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Getahun Abate,1 Daniel F Hoft1,2 1Department of Internal Medicine, Division of Infectious Diseases, Allergy and Immunology, 2Department of Molecular Microbiology and Immunology, Saint Louis University, St. Louis, MO, USA Abstract: Tuberculosis (TB) is still a major global health problem. A third of the world's population is infected with Mycobacterium tuberculosis. Only ~10% of infected individuals develop TB but there are 9 million TB cases with 1.5 million deaths annually. The standard prophylactic treatment regimens for latent TB infection take 3–9 months, and new cases of TB require at least 6 months of treatment with multiple drugs. The management of latent TB infection and TB has become more challenging because of the spread of multidrug-resistant and extremely drug-resistant TB. Intensified efforts to find new TB drugs and immunotherapies are needed. Immunotherapies could modulate the immune system in patients with latent TB infection or active disease, enabling better control of M. tuberculosis replication. This review describes several types of potential immunotherapies with a focus on those which have been tested in humans. Keywords: tuberculosis, HDT, immunotherapy, treatment

Background: One strategy for improving anti-tuberculosis (TB) vaccination involves the use of recombinant bacille Calmette-Guérin (rBCG) overexpressing protective TB antigens. rBCG30, which overexpresses the Mycobacterium tuberculosis secreted antigen Ag85b, was the first rBCG shown to induce significantly greater protection against TB in animals than parental BCG.Methods: We report here the first double-blind phase 1 trial of rBCG30 in 35 adults randomized to receive either rBCG30 or parental Tice BCG intradermally. Clinical reactogenicity was assessed, and state-of-the-art immunological assays were used to study Ag85b-specific immune responses induced by both vaccines.Results: Similar clinical reactogenicity occurred with both vaccines. rBCG30 induced significantly increased Ag85b-specific T cell lymphoproliferation, interferon (IFN)-gamma secretion, IFN-gamma enzyme-linked immunospot responses, and direct ex vivo intracellular IFN-gamma responses. Additional flow cytometry studies measuring carboxyfluorescein succinimidyl ester dilution and intracellular cytokine production demonstrated that rBCG30 significantly enhanced the population of Ag85b-specific CD4(+) and CD8(+) T cells capable of concurrent expansion and effector function. More importantly, rBCG30 significantly increased the number of Ag85b-specific T cells capable of inhibiting intracellular mycobacteria.Conclusions: These results provide proof of principal that rBCG can safely enhance human TB immunity and support further development of rBCG overexpressing Ag85b for TB vaccination. [ABSTRACT FROM AUTHOR]

A third of the world's population is infected with Mycobacterium tuberculosis, and 2 million people die from tuberculosis every year even though the bacille Calmette Guérin (BCG) vaccine has been available for more than 75 years. In order to reduce the immense burden of tuberculosis, new vaccines or vaccination strategies, or both, are urgently needed. Why BCG vaccination has not reduced disease prevalence, especially in the developing world, is not yet understood. Important contributing factors might include background immunity induced by non-tuberculous environmental mycobacteria, diversity of BCG strains, and overattenuation of presently used strains. This review provides a summary of the immune responses thought to be important for protective tuberculosis immunity; various mycobacterial antigens that seem to be promising targets for vaccine-induced immunity; different vaccination approaches being developed for use in people; and the key issues involved in the selection of new vaccines for expanded phase II or III testing. [ABSTRACT FROM AUTHOR]

We developed a carboxyfluorescein succinimidyl ester (CFSE)-based flow-cytometric assay that can detect different subsets of vaccinia-specific T cells capable of both antigen-specific expansion and protective effector functions. Proliferation and effector functions were detected by CFSE dilution and intracellular staining, respectively. Absolute numbers of CD4(+)/CFSE(lo)/interferon (IFN)- gamma (+), CD8(+)/CFSE(lo)/IFN- gamma (+), CD8(+)/CFSE(lo)/granzyme A(+), and CD8(+)/CFSE(lo)/CD107a(+) T cells present after in vitro stimulation with live vaccinia were significantly higher in immunized individuals (P<.05). These CD4(+) and CD8(+) T cell increases were >2 log higher than increases detectable by standard lymphoproliferation and cytotoxicity assays. Vaccinia-specific CD8(+)/CFSE(lo)/IFN- gamma (+) and granzyme A(+) T cell responses were significantly correlated with the results of standard (51)Cr-release cytolytic assays (P<.05). Furthermore, vaccinia induced antigen-specific memory gamma delta T cells. We demonstrate that vaccinia induces robust memory effector CD4(+), CD8(+), and gamma delta T cells, all of which are relevant for protection against smallpox. CFSE-based flow-cytometric assays will be useful in evaluating cell-mediated immune responses induced by new smallpox vaccines. Copyright © 2005 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Intranasal M2SR (M2-deficient Single Replication influenza virus) vaccine induces robust immune responses in animal models and human subjects. A high-throughput multiplexed platform was used to analyze hemagglutinin-specific mucosal antibody responses in adults after a single dose of H3N2 M2SR. Nasal swab specimens were analyzed for total and hemagglutinin-specific IgA. Significant, dose-dependent increases in mucosal antibody responses to vaccine-matched and drifted H3N2 hemagglutinin were observed in M2SR vaccinated subjects regardless of baseline serum and mucosal immune status. These data suggest that M2SR induces broadly cross-reactive mucosal immune responses which may provide better protection against drifted and newly emerging influenza strains., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Immune evasion of Mycobacterium tuberculosis (Mtb) facilitates intracellular bacterial growth. The mechanisms of immune evasion, however, are still not fully understood. In this study, we reveal that tristetraprolin (TTP), one of the best characterized RNA-binding proteins controlling the stability of targeted mRNAs, mediates innate immune evasion of mycobacteria. We found that TTP knockout mice displayed reduced bacterial burden in the early stage after Mtb aerosol challenge. Macrophages deficient in TTP also showed an inhibition in intracellular mycobacterial growth. Live mycobacteria induced TTP protein expression in macrophages, which was blocked by the mTOR inhibitor rapamycin. Rapamycin and AZD8055 specifically blocked 4EBP1 phosphorylation in infected macrophages and suppressed intracellular BCG growth. Rapamycin promoted TTP protein degradation through the ubiquitination pathway, whereas the proteasome inhibitor MG-132 blocked rapamycin function and thus stabilized TTP protein. TTP induction suppressed the expression of iNOS/TNF-α/IL-12/IL-23, and weakened protective immune responses in macrophages, whereas rapamycin enhanced the bactericidal effects through TTP inhibition. Moreover, blocking TTP binding increased the expression of TNF-α and iNOS and suppressed intracellular mycobacterial growth. Overall, our study reveals a novel role for RNA-binding protein TTP in Mtb immune evasion mechanisms and provides a potential target for host-directed therapy against tuberculosis (TB)., (© 2024 The Authors FASEB BioAdvances published by The Federation of American Societies for Experimental Biology.)

Background: A controlled human infection model for assessing tuberculosis (TB) immunity can accelerate new vaccine development., Methods: In this phase 1 dose escalation trial, 92 healthy adults received a single intradermal injection of 2 × 106 to 16 × 106 colony-forming units of Bacillus Calmette-Guérin (BCG). The primary endpoints were safety and BCG shedding as measured by quantitative polymerase chain reaction, colony-forming unit plating, and MGIT BACTEC culture., Results: Doses up to 8 × 106 were safe, and there was evidence for increased BCG shedding with dose escalation. The MGIT time-to-positivity assay was the most consistent and precise measure of shedding. Power analyses indicated that 10% differences in MGIT time to positivity (area under the curve) could be detected in small cohorts (n = 30). Potential biomarkers of mycobacterial immunity were identified that correlated with shedding. Transcriptomic analysis uncovered dose- and time-dependent effects of BCG challenge and identified a putative transcriptional TB protective signature. Furthermore, we identified immunologic and transcriptomal differences that could represent an immune component underlying the observed higher rate of TB disease incidence in males., Conclusions: The safety, reactogenicity, and immunogenicity profiles indicate that this BCG human challenge model is feasible for assessing in vivo TB immunity and could facilitate the vaccine development process., Clinical Trials Registration: NCT01868464 (ClinicalTrials.gov)., Competing Interests: Potential conflicts of interest. D. F. H. receives personal fees for scientific advisory board service for Moderna and Poolbeg Pharma; M. J. M. performs laboratory research and holds clinical trials contracts with Lilly, Pfizer, and Sanofi and receives personal fees for scientific advisory board service from Merck, Meissa Vaccines, Inc, and Pfizer; N. G. R. receives funding from Merck, Sanofi Pasteur, Pfizer, Lilly, and Quidel to perform clinical research and serves as a safety consultant for ICON and Emmes LCC. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete granzyme A (GzmA) protective against intracellular Mtb growth. However, GzmA-enzymatic activity is unnecessary for pathogen inhibition, and the mechanisms of GzmA-mediated protection remain unknown. We show that GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes, and subsequent pathogen clearance within the phagolysosome. Although monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Without access to surface-expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1 was associated with inhibitory activity. Furthermore, GzmA colocalized with and was regulated by protein disulfide isomerase AI (PDIA1), which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify a previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis, and elimination of Mtb in human monocytes, and they highlight PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease., Competing Interests: Potential conflicts of interest. All authors declare that they have no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Influenza A (H7N9) has caused multiple disease waves with evidence of strain diversification. Optimal influenza A (H7N9) prime-boost vaccine strategies are unknown., Methods: We recruited participants who had received monovalent inactivated A/Shanghai/2/2013 (H7N9) vaccine (MIV) approximately 5 years earlier, as follows: MIV with MF59 (MF59 × 2 group), MIV with AS03 (AS03 × 2 group), unadjuvanted MIV (No Adj group), MIV with MF59 or AS03 followed by unadjuvanted MIV (Adjx1 group), and A/H7-naive (unprimed group). Participants were randomized to receive 1 dose of AS03-adjuvanted or unadjuvanted A/Hong Kong/125/2017 (H7N9) MIV and were followed for safety and immunogenicity using hemagglutination inhibition (HAI) and neutralizing antibody assays., Results: We enrolled 304 participants: 153 received the adjuvanted boost and 151 received the unadjuvanted boost. At 21 days postvaccination, the proportion of participants with HAI antibody titers against the boosting vaccine strain of ≥40 in the adjuvanted and unadjuvanted arms, respectively, were 88% and 49% in MF59 × 2 group, 89% and 75% in AS03 × 2 group, 59% and 20% in No Adj group, 94% and 55% in Adjx1group, and 9% and 11% in unprimed group., Conclusions: Serologic responses to a heterologous A(H7N9) MIV boost were highest in participants primed and boosted with adjuvant-containing regimens., Clinical Trials Registration: NCT03738241., Competing Interests: Potential conflicts of interest . I. Y.’s institution received funding to conduct clinical research unrelated to this manuscript from Moderna and Pfizer; and I. Y. consults for Merck and Sanofi-Pasteur. P. W. has received research funding unrelated to this manuscript from Pfizer and Sanofi; and has consulted for Pfizer. C. B. C. serves as a consultant to Pfizer, Moderna, GSK, and Vir. K. M. E. serves as consultant to Bionet and IBM; and member of the Data Safety and Monitoring Boards for Sanofi, X-4 Pharma, Seqirus, Moderna, Pfizer, Merck, and Roche. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Tuberculosis remains an international health threat partly because of limited protection from pulmonary tuberculosis provided by standard intradermal vaccination with Bacillus of Calmette and Guérin (BCG); this may reflect the inability of intradermal vaccination to optimally induce pulmonary immunity. In contrast, respiratory Mycobacterium tuberculosis infection usually results in the immune-mediated bacillary containment of latent tuberculosis infection (LTBI). Here we present RNA-Seq-based assessments of systemic and pulmonary immune cells from LTBI participants and recipients of intradermal and oral BCG. LTBI individuals uniquely display ongoing immune activation and robust CD4 T cell recall responses in blood and lung. Intradermal BCG is associated with robust systemic immunity but only limited pulmonary immunity. Conversely, oral BCG induces limited systemic immunity but distinct pulmonary responses including enhanced inflammasome activation potentially associated with mucosal-associated invariant T cells. Further, IL-9 is identified as a component of systemic immunity in LTBI and intradermal BCG, and pulmonary immunity following oral BCG., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Zika virus (ZIKV) is a flavivirus primarily transmitted by Aedes species mosquitoes, first discovered in Africa in 1947, that disseminated through Southeast Asia and the Pacific Islands in the 2000s. The first ZIKV infections in the Americas were identified in 2014, and infections exploded through populations in Brazil and other countries in 2015/16. ZIKV infection during pregnancy can cause severe brain and eye defects in offspring, and infection in adults has been associated with higher risks of Guillain-Barré syndrome. We initiated a study to describe the natural history of Zika (the disease) and the immune response to infection, for which some results have been reported. In this paper, we identify ZIKV-specific CD4+ and CD8+ T cell epitopes that induce responses during infection. Two screening approaches were utilized: an untargeted approach with overlapping peptide arrays spanning the entire viral genome, and a targeted approach utilizing peptides predicted to bind human MHC molecules. Immunoinformatic tools were used to identify conserved MHC class I supertype binders and promiscuous class II binding peptide clusters predicted to bind 9 common class II alleles. T cell responses were evaluated in overnight IFN-γ ELISPOT assays. We found that MHC supertype binding predictions outperformed the bulk overlapping peptide approach. Diverse CD4+ T cell responses were observed in most ZIKV-infected participants, while responses to CD8+ T cell epitopes were more limited. Most individuals developed a robust T cell response against epitopes restricted to a single MHC class I supertype and only a single or few CD8+ T cell epitopes overall, suggesting a strong immunodominance phenomenon. Noteworthy is that many epitopes were commonly immunodominant across persons expressing the same class I supertype. Nearly all of the identified epitopes are unique to ZIKV and are not present in Dengue viruses. Collectively, we identified 31 immunogenic peptides restricted by the 6 major class I supertypes and 27 promiscuous class II epitopes. These sequences are highly relevant for design of T cell-targeted ZIKV vaccines and monitoring T cell responses to Zika virus infection and vaccination., Competing Interests: MM reported laboratory research and clinical trials contract funding with Lilly, Pfizer, and Sanofi; and personal fees for Scientific Advisory Board service from Merck, Meissa Vaccines, Inc. and Pfizer. Authors FT, AG, WM, LM, and ADG are (or were) employed by EpiVax, Inc, and author CF was employed by The Emmes Company, LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Eickhoff, Meza, Terry, Colbert, Blazevic, Gutiérrez, Stone, Brien, Pinto, El Sahly, Mulligan, Rouphael, Alcaide, Tomashek, Focht, Martin, Moise, De Groot and Hoft.)

Background: We evaluated the associations between baseline influenza virus-specific hemagglutination inhibition (HAI) and microneutralization (MN) titers and subsequent symptomatic influenza virus infection in a controlled human infection study., Methods: We inoculated unvaccinated healthy adults aged 18-49 years with an influenza A/California/04/2009/H1N1pdm-like virus (NCT04044352). We collected serial safety labs, serum for HAI and MN, and nasopharyngeal swabs for reverse-transcription polymerase chain reaction (RT-PCR) testing. Analyses used the putative seroprotective titer of ≥40 for HAI and MN. The primary clinical outcome was mild-to-moderate influenza disease (MMID), defined as ≥1 postchallenge positive qualitative RT-PCR test with a qualifying symptom/clinical finding., Results: Of 76 participants given influenza virus challenge, 54 (71.1%) experienced MMID. Clinical illness was generally very mild. MMID attack rates among participants with baseline titers ≥40 by HAI and MN were 64.9% and 67.9%, respectively, while MMID attack rates among participants with baseline titers <40 by HAI and MN were 76.9% and 78.3%, respectively. The estimated odds of developing MMID decreased by 19% (odds ratio, 0.81 [95% confidence interval, .62-1.06]; P = .126) for every 2-fold increase in baseline HAI. There were no significant adverse events., Conclusions: We achieved a 71.1% attack rate of MMID. High baseline HAI and MN were associated with protection from illness. Clinical Trials Registration. NCT04044352., Competing Interests: Potential conflicts of interest. J. R. O. reports grants to his institution from the National Science Foundation, Bill & Melinda Gates Foundation, Pfizer, NIH, and World Health Organization; consulting fees from Putnam; and participation on advisory boards for Pfizer, Seqirus, and Moderna, all outside the submitted work. D. I. B. reports consulting fees from Rational Vaccines and participation on an advisory board with Moderna and Dynavax, outside the submitted work. C. W. W. reports grants to his institution from Janssen and AstraZeneca, outside the submitted work. M. T. M. reports grants to his institution from NIH, outside the submitted work. R. W. F. reports grants to his institution from Pfizer, outside the submitted work, and clinical trial support from Pfizer. E. W. reports grants to his institution from Clinetic, Moderna, Pfizer, Seqirus, and Iliad Biotechnologies; consulting fees from Iliad Biotechnologies; and participation on the Vaxcyte advisory board. W. A. K. reports grants to her institution from Leidos, outside the submitted work; clinical trial support from Pfizer; subcontract from Leidos; and advisory board participation with the NIH. N. R. reports grants to her institution from Merck, Sanofi Pasteur, Quidel, Pfizer, and Lilly, outside the submitted work. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (Published by Oxford University Press on behalf of Infectious Diseases Society of America 2023.)

Influenza, tetanus, diphtheria, and herpes zoster (HZ) vaccination received within 10 years of the COVID-19 pandemic have been associated with less severe COVID-19 infection. We expanded on this evidence to determine if a receiving two different vaccinations (i.e., HZ and tetanus, diphtheria, and pertussis (Tdap)) was associated with a lower risk for COVID-19 hospitalization. De-identified medical record data from a large mid-western health care system was used to determine if, compared to those with neither HZ or Tdap vaccination, patients with either HZ or Tdap and patients with both HZ and Tdap vaccination had lower risk for COVID-19 hospitalization between 4/1/2020 and 12/31/2020. Confounding was controlled using entropy balancing. Patients (n = 363,293) were 71.5 (±8.4) years of age, 57.8% female and 89.2% White race. Prior to controlling for confounding, as compared to patients without either vaccination, those that had either HZ or Tdap were significantly less likely to have a COVID-19 hospitalization (RR = 0.85; 95 %CI: 0.75-0.95). The risk for hospitalization decreased further among those with both HZ and Tdap vaccination (RR = 0.45; 95 %CI:0.28-0.71). After controlling for confounding, including healthy patient bias, receiving both vs. neither vaccinations remained significantly associated with a lower risk of COVID-19 hospitalization (RR = 0.48; 95 %CI: 0.26-0.90). Receiving both Tdap and HZ vaccination is associated with lower risk for COVID-19 hospitalization. Whether there is any benefit of past vaccination exposure in COVID-19 vaccinated patients should be investigated., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Alternative delivery routes of the current Mycobacterium tuberculosis (Mtb) vaccine, intradermally (ID) delivered BCG, may provide better protection against tuberculosis, and be more easily administered. Here, we use rhesus macaques to compare the airway immunogenicity of BCG delivered via either ID or intragastric gavage vaccination. Ag-specific CD4 T cell responses in the blood were similar after BCG vaccination via gavage or ID injection. However, gavage BCG vaccination induced significantly lower T cell responses in the airways compared to intradermal BCG vaccination. Examining T cell responses in lymph node biopsies showed that ID vaccination induced T cell priming in skin-draining lymph nodes, while gavage vaccination induced priming in the gut-draining nodes, as expected. While both delivery routes induced highly functional Ag-specific CD4 T cells with a Th1* phenotype (CXCR3 + CCR6 + ), gavage vaccination induced the co-expression of the gut-homing integrin α 4 β 7 on Ag-specific Th1* cells, which was associated with reduced migration into the airways. Thus, in rhesus macaques, the airway immunogenicity of gavage BCG vaccination may be limited by the imprinting of gut-homing receptors on Ag-specific T cells primed in intestinal lymph nodes. IMPORTANCE Mycobacterium tuberculosis (Mtb) is a leading cause of global infectious disease mortality. The vaccine for Mtb, Bacillus Calmette-Guérin (BCG), was originally developed as an oral vaccine, but is now given intradermally. Recently, clinical studies have reevaluated oral BCG vaccination in humans and found that it induces significant T cell responses in the airways. Here, we use rhesus macaques to compare the airway immunogenicity of BCG delivered intradermally or via intragastric gavage. We find that gavage BCG vaccination induces Mtb-specific T cell responses in the airways, but to a lesser extent than intradermal vaccination. Furthermore, gavage BCG vaccination induces the gut-homing receptor a4ß7 on Mtb-specific CD4 T cells, which was associated with reduced migration into the airways. These data raise the possibility that strategies to limit the induction of gut-homing receptors on responding T cells may enhance the airway immunogenicity of oral vaccines.

Introduction: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection is a serious threat to human health. Vaccination with BCG prevents the development of the most severe forms of TB disease in infants and was recently shown to prevent Mtb infection in previously uninfected adolescents. γδ T cells play a major role in host defense at mucosal sites and are known to respond robustly to mycobacterial infection. However, our understanding of the effects of BCG vaccination on γδ T cell responses is incomplete., Methods: In this study we performed γδ T cell receptor (TCR) repertoire sequencing of samples provided pre- and post-BCG vaccination from 10 individuals to identify specific receptors and TCR clones that are induced by BCG., Results: Overall, there was no change in the diversity of γTCR or δTCR clonotypes in post- vs pre-BCG samples. Furthermore, the frequencies of TCR variable and joining region genes were minimally modulated by BCG vaccination at either the γTCR or δTCR loci. However, the γTCR and δTCR repertoires of individuals were highly dynamic; a median of ~1% of γTCR and ~6% of δTCR in the repertoire were found to significantly expand or contract in post- vs pre-BCG comparisons (FDR-q < 0.05). While many of the clonotypes whose frequency changed after BCG vaccination were not shared among multiple individuals in the cohort, several shared (i.e., "public") clonotypes were identified with a consistent increase or decrease in frequency across more than one individual; the degree of sharing of these clonotypes was significantly greater than the minimal sharing that would be expected among γTCR and δTCR repertoires. An in vitro analysis of Mtb antigen-reactive γδ T cells identified clonotypes that were similar or identical to the single-chain γTCRs and δTCRs that changed consistently after BCG vaccination; pairings of γTCRs and δTCRs that increased after BCG vaccination were significantly over-represented among the Mtb-reactive γδ T cells (p = 1.2e-6)., Discussion: These findings generate hypotheses about specific γδTCR clonotypes that may expand in response to BCG vaccination and may recognize Mtb antigens. Future studies are required to validate and characterize these clonotypes, with an aim to better understand the role of γδ T cells in Mtb immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Xia, Blazevic, Fiore-Gartland and Hoft.)

Adjuvant-containing subunit vaccines represent a promising approach for protection against tuberculosis (TB), but current candidates require refrigerated storage. Here we present results from a randomized, double-blinded Phase 1 clinical trial (NCT03722472) evaluating the safety, tolerability, and immunogenicity of a thermostable lyophilized single-vial presentation of the ID93 + GLA-SE vaccine candidate compared to the non-thermostable two-vial vaccine presentation in healthy adults. Participants were monitored for primary, secondary, and exploratory endpoints following intramuscular administration of two vaccine doses 56 days apart. Primary endpoints included local and systemic reactogenicity and adverse events. Secondary endpoints included antigen-specific antibody (IgG) and cellular immune responses (cytokine-producing peripheral blood mononuclear cells and T cells). Both vaccine presentations are safe and well tolerated and elicit robust antigen-specific serum antibody and Th1-type cellular immune responses. Compared to the non-thermostable presentation, the thermostable vaccine formulation generates greater serum antibody responses (p < 0.05) and more antibody-secreting cells (p < 0.05). In this work, we show the thermostable ID93 + GLA-SE vaccine candidate is safe and immunogenic in healthy adults., (© 2023. The Author(s).)

Introduction: Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. The goal of this study was to benchmark key technical and analytical parameters of RNA sequencing (RNA-seq) in the context of a multi-site, double-blind randomized vaccine clinical trial., Methods: We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects before and after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two replicate datasets (5 time points for 50 samples each). We evaluated the impact of (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data and established the impact of sequencing depth and gene filtering on transcriptome representation. Lastly, we modeled statistical power to detect DEGs for a range of sample sizes, effect sizes, and sequencing depths., Results and Discussion: Our results showed that (i) filtering lowly-expressed genes is recommended to improve fold-change accuracy and inter-site agreement, if possible guided by mRNA spike-ins (ii) read length did not have a major impact on DEG detection, (iii) applying fold-change cutoffs for DEG detection reduced inter-set agreement and should be used with caution, if at all, (iv) reduction in sequencing depth had a minimal impact on statistical power but reduced the identifiable fraction of the PBMC transcriptome, (v) after sample size, effect size (i.e. the magnitude of fold change) was the most important driver of statistical power to detect DEG. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies., Competing Interests: JG, TLJ, TG, CEG, KJS were employed by The Emmes Company. EA has consulted for Pfizer, Sanofi Pasteur, GSK, Janssen, Moderna, and Medscape, and his institution receives funds to conduct clinical research unrelated to this manuscript from MedImmune, Regeneron, PaxVax, Pfizer, GSK, Merck, Sanofi-Pasteur, Janssen, and Micron. He also serves on a safety monitoring board for Kentucky BioProcessing, Inc. and Sanofi Pasteur. He serves on a data adjudication board for WCG and ACI Clinical. His institution has also received funding from NIH to conduct clinical trials of COVID-19 vaccines. MM reported potential competing interests: laboratory and clinical trial contract funding for vaccines or MAB vs SARS-CoV-2 with Lilly, Pfizer, and Sanofi; personal fees for Scientific Advisory Board service from Merck, Meissa Vaccines, Inc., and Pfizer. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MG declared a shared affiliation with the author RH to the handling editor at the time of review., (Copyright © 2023 Goll, Bosinger, Jensen, Walum, Grimes, Tharp, Natrajan, Blazevic, Head, Gelber, Steenbergen, Patel, Sanz, Rouphael, Anderson, Mulligan and Hoft.)

Introduction: Trypanosoma cruzi is the protozoan parasite causing Chagas disease, a Neglected Tropical Disease that affects 8 million people and causes 12,000 deaths per year, primarily because of cardiac pathology. Effective vaccination for T. cruzi remains an elusive goal. The use of a live vaccine vector, especially one that mimics the pathogen target, may be superior to the use of recombinant protein or DNA vaccine formulations., Methods: We generated recombinant Leishmania major , a related trypanosomatid parasite, as a vaccine vehicle to express the immunogenic T. cruzi trans -sialidase (TS) antigen. The induction of T cell and antibody responses, as well as T. cruzi protective immunity generated by these vaccines were assessed in vivo., Results: We demonstrate that mice inoculated with these recombinant TS-expressing L. major parasites mount T cell and antibody responses directed against TS and are protected against future T. cruzi infection. We also show that the partially attenuated dhfr-ts- CC1 L . major strain, previously found to induce protective immunity to virulent L. major infection without causing pathology, can also be engineered to express the TS antigen. This latter recombinant may represent a safe and effective option to explore for ultimate use in humans., Discussion: Altogether, these data indicate that L. major can stably express a T. cruzi antigen and induce T. cruzi -specific protective immunity, warranting further investigation of attenuated Leishmania parasites as vaccine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cai, O’Shea, Eickhoff, Guo, Lewis, Beverley and Hoft.)

Plasmalogens are plasma-borne antioxidant phospholipid species that provide protection as cellular lipid components during cellular oxidative stress. In this study we investigated plasma plasmalogen levels in human sepsis as well as in rodent models of infection. In humans, levels of multiple plasmenylethanolamine molecular species were decreased in septic patient plasma compared to control subject plasma as well as an age-aligned control subject cohort. Additionally, lysoplasmenylcholine levels were significantly decreased in septic patients compared to the control cohorts. In contrast, plasma diacyl phosphatidylethanolamine and phosphatidylcholine levels were elevated in septic patients. Lipid changes were also determined in rats subjected to cecal slurry sepsis. Plasma plasmenylcholine, plasmenylethanolamine, and lysoplasmenylcholine levels were decreased while diacyl phosphatidylethanolamine levels were increased in septic rats compared to control treated rats. Kidney levels of lysoplasmenylcholine as well as plasmenylethanolamine molecular species were decreased in septic rats. Interestingly, liver plasmenylcholine and plasmenylethanolamine levels were increased in septic rats. Since COVID-19 is associated with sepsis-like acute respiratory distress syndrome and oxidative stress, plasmalogen levels were also determined in a mouse model of COVID-19 (intranasal inoculation of K18 mice with SARS-CoV-2). 3 days following infection, lung infection was confirmed as well as cytokine expression in the lung. Multiple molecular species of lung plasmenylcholine and plasmenylethanolamine were decreased in infected mice. In contrast, the predominant lung phospholipid, dipalmitoyl phosphatidylcholine, was not decreased following SARS-CoV-2 infection. Additionally total plasmenylcholine levels were decreased in the plasma of SARS-CoV-2 infected mice. Collectively, these data demonstrate the loss of plasmalogens during both sepsis and SARS-CoV-2 infection. This study also indicates plasma plasmalogens should be considered in future studies as biomarkers of infection and as prognostic indicators for sepsis and COVID-19 outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pike, McGuffee, Geerling, Albert, Hoft, Shashaty, Meyer, Pinto and Ford.)

Background: Multiple types of vaccinations are associated with lower risk for dementia, but it is not known if receiving more than one vaccination type is associated with a greater decrease in incident dementia as compared with receiving only one type. We determined if dementia risk is lowest in patients who receive both herpes zoster (HZ) and tetanus, diphtheria, pertussis (Tdap) vaccinations as compared with receipt of only one or the other type of vaccination., Methods: Primary analysis in a Veterans Health Administration (VA) cohort was replicated in private sector medical claims data. Eligible patients were ≥65 years of age and free of dementia for 2 years prior to baseline (VHA n = 80,070; MarketScan n = 129,200). At index, patients either had both HZ and Tdap, only HZ, only Tdap, or neither vaccination. Confounding was controlled with generalized boosted propensity scores and inverse probability of treatment weighting. Competing risk (VHA) and Cox proportional hazard (MarketScan) models estimated the association between vaccination status and incident dementia., Results: VHA patients' mean age was 76.8 ± 7.6 years, 4.4% were female and 90.9% were White, and MarketScan patients' mean age was 70.5 ± 5.9 and 65.4% were female. In both cohorts, having both HZ and Tdap vaccinations compared with no vaccination was significantly associated with lower dementia risk (VHA HR = 0.50; 95% CI: 0.43-0.59; MarketScan HR = 0.58; 95% CI: 0.38-0.89). In both cohorts, compared with neither vaccination, patients with only one or the other vaccination types had a significantly lower risk for dementia. Incident dementia was lower in patients with both vaccinations versus only one vaccination type., Conclusions and Relevance: Receiving two types of vaccinations versus one type was associated with lower dementia risk. Vaccinations may have non-specific associations with incident dementia. Low cost and accessible, common adult vaccinations may be an overlooked intervention for reducing dementia risk., (© 2021 The Authors. Journal of the American Geriatrics Society published by Wiley Periodicals LLC on behalf of The American Geriatrics Society.)

Cytotoxic lymphocytes release proteins contained within the cytoplasmic cytolytic granules after recognition of infected or tumor target cells. These cytotoxic granular proteins (namely granzymes, granulysin, and perforin) are key immunological mediators within human cellular immunity. The availability of highly purified cytotoxic proteins has been fundamental for understanding their function in immunity and mechanistic involvement in sepsis and autoimmunity. Methods for recovery of native cytotoxic proteins can be problematic leading to: 1) the co-purification of additional proteins, confounding interpretation of function, and 2) low yields of highly purified proteins. Recombinant protein expression of individual cytolytic components can overcome these challenges. The use of mammalian expression systems is preferred for optimal post-translational modifications and avoidance of endotoxin contamination. Some of these proteins have been proposed for host directed human therapies (e.g. - granzyme A), or treatment of systemic infections or tumors as in granulysin. We report here a novel expression system using HEK293T cells for cost-effective purification of high yields of human granzymes (granzyme A and granzyme B) and granulysin with enhanced biological activity than previous reports. The resulting proteins are free of native contaminants, fold correctly, and remain enzymatically active. Importantly, these improvements have also led to the first purification of biologically active recombinant human granulysin in high yields from a mammalian system. This method can be used as a template for purification of many other secreted cellular proteins and may lead to advances for human medicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rasi, Hameed, Matthey, Bera, Grandgenett, Salentinig, Walch and Hoft.)

Background: Non-protein antigen classes can be presented to T cells by near-monomorphic antigen-presenting molecules such as CD1, MR1, and butyrophilin 3A1. Such T cells, referred to as donor unrestricted T (DURT) cells, typically express stereotypic T cell receptors. The near-unrestricted nature of DURT cell antigen recognition is of particular interest for vaccine development, and we sought to define the roles of DURT cells, including MR1-restricted MAIT cells, CD1b-restricted glucose monomycolate (GMM)-specific T cells, CD1d-restricted NKT cells, and γδ T cells, in vaccination against Mycobacterium tuberculosis., Methods: We compared and characterized DURT cells following primary bacille Calmette-Guerin (BCG) vaccination in a cohort of vaccinated and unvaccinated infants, as well as before and after BCG-revaccination in adults., Findings: BCG (re)vaccination did not modulate peripheral blood frequencies, T cell activation or memory profiles of MAIT cells, CD1b-restricted GMM-specific and germline-encoded mycolyl-reactive (GEM) cells or CD1d-restricted NKT cells. By contrast, primary BCG vaccination was associated with increased frequencies of γδ T cells as well as a novel subset of CD26 + CD161 + TRAV1-2 - IFN-γ-expressing CD4 + T cells in infants., Interpretation: Our findings, that most DURT cell populations were not modulated by BCG, do not preclude a role of BCG in modulating other qualitative aspects of DURT cells. More studies are required to understand the full potential of DURT cells in new TB vaccine strategies., Funding: Aeras, the National Institutes of Health, and the Bill and Melinda Gates Foundation., Competing Interests: Declaration of interests No competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Recent reports indicate that African Americans have higher mortality rates from SARS-CoV-2 coronavirus disease 19 (COVID-19) compared to Caucasians, with more marked differences in the Midwest region of the US. This study was performed to study differences in COVID-19 related mortality and hospital length of stay (LOS) between African Americans and Caucasians in Midwest setting, and identify factors associated with mortality and LOS., Methods: Data were collected from the electronic health records (EHR) of patients admitted to hospitals in Midwest region of the US. EHR of 471 COVID-19 patients were reviewed., Results: Approximately 63% were African Americans and 34% Caucasians. One hundred sixteen variables were tested. There was no significant difference in hospital mortality between African Americans and Caucasians (OR 1, 95% CI 0.48-1.94). Older age, Chronic kidney disease, mental status change, mechanical ventilation, vasopressor support, high neutrophil count, elevated AST and ALT, high lung involvement severity score and elevated CRP were associated with mortality in a univariate analysis (P < 0.05). Multivariable modeling indicated that mechanical ventilation was the only factor that predicted mortality (OR 6, 95% CI: 2.94-12.48). The LOS did not differ in African Americans and Caucasians. The use of oxygen via high flow nasal cannula (Survival Estimate 1.6, 95% CI: 1.20-2.26), low estimated glomerular filtration rate (Survival Estimate 1.4, 95% CI: 1.05-1.82) and mechanical ventilation (Survival Estimate 3.5, 95% CI: 2.72-4.37) were predictors of LOS., Conclusion: This study performed in Midwest setting in the US showed that race did not affect in-hospital mortality and LOS. Our analysis demonstrated new predictors of LOS., (Copyright © 2021. Published by Elsevier Inc.)

Metabolic reprogramming of cancer cells creates a unique tumor microenvironment (TME) characterized by the limited availability of nutrients, which subsequently affects the metabolism, differentiation, and function of tumor-infiltrating T lymphocytes (TILs). TILs can also be inhibited by tumor-derived metabolic waste products and low oxygen. Therefore, a thorough understanding of how such unique metabolites influence mammalian T cell differentiation and function can inform novel anticancer therapeutic approaches. Here, we highlight the importance of these metabolites in modulating various T cell subsets within the TME, dissecting how these changes might alter clinical outcomes. We explore potential TME metabolic determinants that might constitute candidate targets for cancer immunotherapies, ideally leading to future strategies for reprogramming tumor metabolism to potentiate anticancer T cell functions., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Metabolic disorder is one of the hallmarks of cancers, and reprogramming of metabolism is becoming a novel strategy for cancer treatment. Citrate is a key metabolite and critical metabolic regulator linking glycolysis and lipid metabolism in cellular energy homeostasis. Here it is reported that citrate treatment (both sodium citrate and citric acid) significantly suppresses tumor cell proliferation and growth in various tumor types. Mechanistically, citrate promotes excessive lipid biosynthesis and induces disruption of lipid metabolism in tumor cells, resulting in tumor cell senescence and growth inhibition. Furthermore, ATM-associated DNA damage response cooperates with MAPK and mTOR signaling pathways to control citrate-induced tumor cell growth arrest and senescence. In vivo studies further demonstrate that citrate administration dramatically inhibits tumor growth and progression in a colon cancer xenograft model. Importantly, citrate administration combined with the conventional chemotherapy drugs exhibits synergistic antitumor effects in vivo in the colon cancer models. These results clearly indicate that citrate can reprogram lipid metabolism and cell fate in cancer cells, and targeting citrate can be a promising therapeutic strategy for tumor treatment., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Plasmalogens are a class of phospholipids containing vinyl ether linked aliphatic groups at the sn-1 position. Plasmalogens are known to contain 16- and 18-carbon aliphatic groups at the sn-1 position. Here, we reveal that the human neutrophil plasmenylethanolamine pool uniquely includes molecular species with very long carbon chain (VLC) aliphatic groups, including 20-, 22- and 24-carbon vinyl ether linked aliphatic groups at the sn-1 position. We identified these novel VLC plasmalogen species by electrospray ionization mass spectrometry methods. VLC plasmalogens were only found in the neutrophil plasmenylethanolamine pool. During neutrophil activation, VLC plasmenylethanolamines undergo myeloperoxidase-dependent oxidation to produce VLC 2-chlorofatty aldehyde and its oxidation product, 2-chlorofatty acid (2-ClFA). Furthermore, plasma concentrations of VLC 2-ClFA are elevated in human sepsis. These studies demonstrate for the first time VLC plasmenylethanolamine molecular species, their myeloperoxidase-mediated chlorolipid products and the presence of these chlorolipids in human sepsis., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Herpes zoster (HZ) infection increases dementia risk, but it is not known if herpes zoster vaccination is associated with lower risk for dementia. We determined if HZ vaccination, compared to no HZ vaccination, is associated with lower risk for incident dementia., Methods and Findings: Data was obtained from Veterans Health Affairs (VHA) medical records (10/1/2008-9/30/2019) with replication in MarketScan® commercial and Medicare claims (1/1/2009-12/31/2018). Eligible patients were ≥65 years of age and free of dementia for two years prior to baseline (VHA n = 136,016; MarketScan n = 172,790). Two index periods (either start of 2011 or 2012) were defined, where patients either had or did not have a HZ vaccination. Confounding was controlled with propensity scores and inverse probability of treatment weighting. Competing risk (VHA) and Cox proportional hazard (MarketScan) models estimated the association between HZ vaccination and incident dementia in all patients and in age (65-69, 70-74, ≥75) and race (White, Black, Other) sub-groups. Sensitivity analysis measured the association between HZ vaccination and incident Alzheimer's dementia (AD). HZ vaccination at index versus no HZ vaccination throughout follow-up. VHA patients mean age was 75.7 (SD±7.4) years, 4.0% were female, 91.2% white and 20.2% had HZ vaccination. MarketScan patients mean age was 69.9 (SD±5.7) years, 65.0% were female and 14.2% had HZ vaccination. In both cohorts, HZ vaccination compared with no vaccination, was significantly associated with lower dementia risk (VHA HR = 0.69; 95%CI: 0.67-0.72; MarketScan HR = 0.65; 95%CI:0.57-0.74). HZ vaccination was not related to dementia risk in MarketScan patients aged 65-69 years. No difference in HZ vaccination to dementia effects were found by race. HZ vaccination was associated with lower risk for AD., Conclusions: HZ vaccination is associated with reduced risk of dementia. Vaccination may provide nonspecific neuroprotection by training the immune system to limit damaging inflammation, or specific neuroprotection that prevents viral cytopathic effects., Competing Interests: The authors have declared that no competing interests exist.

Background: Two Taiwan based studies indicated influenza vaccinations are associated with lower risk for dementia in patient cohorts with chronic disease. We determined if such associations exist in a large, nationally distributed sample of U.S. patients not selected for chronic disease., Methods: Data was obtained from Veterans Health Administration medical records (9/1/2009 - 8/31/19). Eligible patients were ≥65 years of age and free of dementia for two years prior to enrollment through the end of the first influenza season (9/1/2009 to 3/1/2012). Competing risk models estimated the risk of dementia in those with influenza vaccination (n = 66,822) compared to those without vaccination (n = 56,925). Propensity scores and inverse probability of treatment weighting controlled for confounding., Results: On average, patients were 75.5 (±7.3) years of age, 3.8% were female and 91.6% were white race. After controlling for confounding, patients with influenza vaccination were significantly less likely to develop dementia compared to patients without vaccination (HR = 0.86; 95 %CI:0.83-0.88). Patients with 1, 2 or 3-5 vaccines vs. none had similar risks for dementia and patients with ≥ 6 influenza vaccines vs. none had a significant lower risk for dementia (HR = 0.88, 95 %CI: 0.83-0.94)., Conclusions: Repeated receipt of influenza vaccinations, compared to remaining unvaccinated, is associated with lower risk for dementia. This is consistent with the hypotheses that vaccinations may reduce risk of dementia by training the immune system and not by preventing specific infectious disease. If vaccines are identified as causative factors in reducing incident dementia, they offer an inexpensive, low-risk intervention with effects greater than any existing preventive measure., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Authors of this work are supported by funding from the Benter Foundation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world's population currently infected. Data indicate that γ 9 δ 2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ 9 δ 2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA's action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rasi, Wood, Eickhoff, Xia, Pozzi, Edwards, Walch, Bovenschen and Hoft.)

Background: Adult vaccinations may reduce risk for dementia. However, it has not been established whether tetanus, diphtheria, pertussis (Tdap) vaccination is associated with incident dementia., Methods: Hypotheses were tested in a Veterans Health Affairs (VHA) cohort and replicated in a MarketScan medical claims cohort. Patients were at least 65 years of age and free of dementia for 2 years prior to index date. Patients either had or did not have a Tdap vaccination by the start of either of the 2 index periods (2011 or 2012). Follow-up continued through 2018. Controls had no Tdap vaccination for the duration of follow-up. Confounding was controlled using entropy balancing. Competing risk (VHA) and Cox proportional hazard (MarketScan) models estimated the association between Tdap vaccination and incident dementia in all patients and age subgroups (65-69, 70-74, and ≥75 years)., Results: VHA patients were, on average, 75.6 (SD ± 7.5) years of age, 4% female, and 91.2% were White. MarketScan patients were 69.8 (SD ± 5.6) years of age, on average and 65.4% were female. After controlling for confounding, patients with, compared to without, Tdap vaccination had a significantly lower risk for dementia in both cohorts (VHA: hazard ratio [HR] = 0.58; 95% confidence interval [CI]:0.54-0.63 and MarketScan: HR = 0.58; 95% CI:0.48-0.70)., Conclusions: Tdap vaccination was associated with a 42% lower dementia risk in 2 cohorts with different clinical and sociodemographic characteristics. Several vaccine types are linked to decreased dementia risk, suggesting that these associations are due to nonspecific effects on inflammation rather than vaccine-induced pathogen-specific protective effects., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Natural killer (NK) and natural killer T (NKT) cells are two important cell subsets of the innate immune system. NK and NKT cells share many phenotypes and functions for anti-tumor immunity; however, the dynamic changes in phenotypes and functional interactions within the tumor microenvironment during tumor development and progression are unknown. Here we report that NK and NKT cells have distinct properties, metabolic profiles, and functions during tumor development. Using the mouse E0771 breast cancer and B16 melanoma models, we found that both NK and NKT cells are dynamically involved in the immune responses to cancer but have distinct distributions and phenotypic profiles in tumor sites and other peripheral organs during the course of tumor development and progression. In the early stages of tumor development, both NK and NKT cells exhibit effector properties. In the later cancer stages, NK and NKT cells have impaired cytotoxic capacities and dysfunctional states. NK cells become senescent cells, while NKT cells, other than invariant NKT (iNKT) cells, are exhausted in the advanced cancers. In contrast, iNKT cells develop increases in activation and effector function within the breast tumor microenvironment. In addition, senescent NK cells have heightened glucose and lipid metabolism, but exhausted NKT cells display unbalanced metabolism in tumor microenvironments of both breast cancer and melanoma tumor models. These studies provide a better understanding of the dynamic and distinct functional roles of NK and NKT cells in anti-tumor immunity, which may facilitate the development of novel immunotherapies targeting NK and NKT cells for cancer treatment.

Trypanosoma cruzi is the intracellular parasite of Chagas disease, a chronic condition characterized by cardiac and gastrointestinal morbidity. Protective immunity requires CD4 + T cells, and Th1 cells and gamma interferon (IFN-γ) are important players in host defense. More recently, Th17 cells and interleukin 17A (IL-17A) have been shown to exert protective functions in systemic T. cruzi infection. However, it remains unclear whether Th17 cells and IL-17A protect in the mucosa, the initial site of parasite invasion in many human cases. We found that IL-17RA knockout (KO) mice are highly susceptible to orogastric infection, indicating an important function for this cytokine in mucosal immunity to T. cruzi. To investigate the specific role of Th17 cells for mucosal immunity, we reconstituted RAG1 KO mice with T. cruzi - specific T cell receptor transgenic Th17 cells prior to orogastric T. cruzi challenges. We found that Th17 cells provided protection against gastric mucosal T. cruzi infection, indicated by significantly lower stomach parasite burdens. In vitro macrophage infection assays revealed that protection by Th17 cells is reduced with IL-17A neutralization or reversed by loss of macrophage NADPH oxidase activity. Consistently with this, mice lacking functional NADPH oxidase were not protected by Th17 cell transfer. These data are the first report that Th17 cells protect against mucosal T. cruzi infection and identify a novel protective mechanism involving the induction of NADPH oxidase activity by IL-17A. These studies provide important insights for Chagas vaccine development and, more broadly, increase our understanding of the diverse roles of Th17 cells in host defense.

The functional state of T cells is a key determinant for effective antitumor immunity and immunotherapy. Cellular metabolism, including lipid metabolism, controls T cell differentiation, survival, and effector functions. Here, we report that development of T cell senescence driven by both malignant tumor cells and regulatory T cells is a general feature in cancers. Senescent T cells have active glucose metabolism but exhibit unbalanced lipid metabolism. This unbalanced lipid metabolism results in changes of expression of lipid metabolic enzymes, which, in turn, alters lipid species and accumulation of lipid droplets in T cells. Tumor cells and T reg cells drove elevated expression of group IVA phospholipase A 2 , which, in turn, was responsible for the altered lipid metabolism and senescence induction observed in T cells. Mitogen-activated protein kinase signaling and signal transducer and activator of transcription signaling coordinately control lipid metabolism and group IVA phospholipase A 2 activity in responder T cells during T cell senescence. Inhibition of group IVA phospholipase A 2 reprogrammed effector T cell lipid metabolism, prevented T cell senescence in vitro, and enhanced antitumor immunity and immunotherapy efficacy in mouse models of melanoma and breast cancer in vivo. Together, these findings identify mechanistic links between T cell senescence and regulation of lipid metabolism in the tumor microenvironment and provide a new target for tumor immunotherapy., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Competing Interests: Authors report no conflict of interest

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response., Competing Interests: The authors have declared that no competing interests exist.

T H 17 cells have been extensively investigated in inflammation, autoimmune diseases, and cancer. The precise molecular mechanisms for T H 17 cell regulation, however, remain elusive, especially regulation at the post-transcriptional level. Tristetraprolin (TTP) is an RNA-binding protein important for degradation of the mRNAs encoding several proinflammatory cytokines. With newly generated T cell-specific TTP conditional knockout mice (CD4 Cre TTP f/f ), we found that aging CD4 Cre TTP f/f mice displayed an increase of IL-17A in serum and spontaneously developed chronic skin inflammation along with increased effector T H 17 cells in the affected skin. TTP inhibited T H 17 cell development and function by promoting IL-17A mRNA degradation. In a DSS-induced colitis model, CD4 Cre TTP f/f mice displayed severe colitis and had more T H 17 cells and serum IL-17A compared with wild-type mice. Furthermore, neutralization of IL-17A reduced the severity of colitis. Our results reveal a new mechanism for regulating T H 17 function and T H 17-mediated inflammation post-transcriptionally by TTP, suggests that TTP might be a novel therapeutic target for the treatment of T H 17-mediated diseases., (Copyright © 2020 Peng, Ning, Wang, Lai, Wei, Stumpo, Blackshear, Fu, Hou, Hoft and Liu.)

Tularemia is a highly infectious and contagious disease caused by the bacterium Francisella tularensis . To better understand human response to a live-attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated, and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we fully characterized individual lipid species within predominant lipid classes to identify changes in the plasma lipidome during the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid classes. These included 5-hydroxyeicosatetraenoic acid (5-HETE) which is indicative of lipoxygenase activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a dihydroxyeicosatrienoic acid (DHET) by day 7 post-vaccination, shedding light on the kinetics of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 16:1 cholesteryl ester, and other glycerophospholipids; several of these changes in abundance were correlated with serum cytokines and T cell activation. These data provide new insights into alterations in plasma lipidome post-tularemia vaccination, potentially identifying key mediators and pathways involved in vaccine response and efficacy.

Francisella tularensis ( F. tularensis ) is an intracellular pathogen that causes a potentially debilitating febrile illness known as tularemia. F. tularensis can be spread by aerosol transmission and cause fatal pneumonic tularemia. If untreated, mortality rates can be as high as 30%. To study the host responses to a live-attenuated tularemia vaccine, peripheral blood mononuclear cell (PBMC) samples were assayed from 10 subjects collected pre- and post-vaccination, using both the 2D-DIGE/MALDI-MS/MS and LC-MS/MS approaches. Protein expression related to antigen processing and presentation, inflammation (PPARγ nuclear receptor), phagocytosis, and gram-negative bacterial infection was enriched at Day 7 and/or Day 14. Protein candidates that could be used to predict human immune responses were identified by evaluating the correlation between proteome changes and humoral and cellular immune responses. Consistent with the proteomics data, parallel transcriptomics data showed that MHC class I and class II-related signals important for protein processing and antigen presentation were up-regulated, further confirming the proteomic results. These findings provide new biological insights that can be built upon in future clinical studies, using live attenuated strains as immunogens, including their potential use as surrogates of protection.

Computational vaccinology includes epitope mapping, antigen selection, and immunogen design using computational tools. Tools that facilitate the in silico prediction of immune response to biothreats, emerging infectious diseases, and cancers can accelerate the design of novel and next generation vaccines and their delivery to the clinic. Over the past 20 years, vaccinologists, bioinformatics experts, and advanced programmers based in Providence, Rhode Island, USA have advanced the development of an integrated toolkit for vaccine design called iVAX, that is secure and user-accessible by internet. This integrated set of immunoinformatic tools comprises algorithms for scoring and triaging candidate antigens, selecting immunogenic and conserved T cell epitopes, re-engineering or eliminating regulatory T cell epitopes, and re-designing antigens to induce immunogenicity and protection against disease for humans and livestock. Commercial and academic applications of iVAX have included identifying immunogenic T cell epitopes in the development of a T-cell based human multi-epitope Q fever vaccine, designing novel influenza vaccines, identifying cross-conserved T cell epitopes for a malaria vaccine, and analyzing immune responses in clinical vaccine studies. Animal vaccine applications to date have included viral infections of pigs such as swine influenza A, PCV2, and African Swine Fever. "Rapid-Fire" applications for biodefense have included a demonstration project for Lassa Fever and Q fever. As recent infectious disease outbreaks underscore the significance of vaccine-driven preparedness, the integrated set of tools available on the iVAX toolkit stand ready to help vaccine developers deliver genome-derived, epitope-driven vaccines., (Copyright © 2020 De Groot, Moise, Terry, Gutierrez, Hindocha, Richard, Hoft, Ross, Noe, Takahashi, Kotraiah, Silk, Nielsen, Minassian, Ashfield, Ardito, Draper and Martin.)

The functional state of the preexisting T cells in the tumor microenvironment is a key determinant for effective antitumor immunity and immunotherapy. Increasing evidence suggests that immunosenescence is an important state of T cell dysfunction that is distinct from exhaustion, a key strategy used by malignant tumors to evade immune surveillance and sustain the suppressive tumor microenvironment. Here, we discuss the phenotypic and functional characteristics of senescent T cells and their role in human cancers. We also explore the possible mechanisms and signaling pathways responsible for induction of T cell senescence by malignant tumors, and then discuss potential strategies to prevent and/or reverse senescence in tumor-specific T cells. A better understanding of these critical issues should provide novel strategies to enhance cancer immunotherapy.

Influenza world-wide causes significant morbidity and mortality annually, and more severe pandemics when novel strains evolve to which humans are immunologically naïve. Because of the high viral mutation rate, new vaccines must be generated based on the prevalence of circulating strains every year. New approaches to induce more broadly protective immunity are urgently needed. Previous research has demonstrated that influenza-specific T cells can provide broadly heterotypic protective immunity in both mice and humans, supporting the rationale for developing a T cell-targeted universal influenza vaccine. We used state-of-the art immunoinformatic tools to identify putative pan-HLA-DR and HLA-A2 supertype-restricted T cell epitopes highly conserved among > 50 widely diverse influenza A strains (representing hemagglutinin types 1, 2, 3, 5, 7 and 9). We found influenza peptides that are highly conserved across influenza subtypes that were also predicted to be class I epitopes restricted by HLA-A2. These peptides were found to be immunoreactive in HLA-A2 positive but not HLA-A2 negative individuals. Class II-restricted T cell epitopes that were highly conserved across influenza subtypes were identified. Human CD4 + T cells were reactive with these conserved CD4 epitopes, and epitope expanded T cells were responsive to both H1N1 and H3N2 viruses. Dendritic cell vaccines pulsed with conserved epitopes and DNA vaccines encoding these epitopes were developed and tested in HLA transgenic mice. These vaccines were highly immunogenic, and more importantly, vaccine-induced immunity was protective against both H1N1 and H3N2 influenza challenges. These results demonstrate proof-of-principle that conserved T cell epitopes expressed by widely diverse influenza strains can induce broadly protective, heterotypic influenza immunity, providing strong support for further development of universally relevant multi-epitope T cell-targeting influenza vaccines., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) remains a tremendous global health problem, with 1/4 of the world's population infected and causing > 1 million deaths annually. Intradermal Bacillus Calmette-Guérin (BCG) vaccine given during infancy protects against severe forms of acute disease but does not prevent chronic infection or development of pulmonary TB. TB vaccine mucosal targeting potentially could induce mucosal resident immune cells with increased protective capacity against pulmonary infection and disease. Sublingual (SL) administration of vaccines may be an optimal mucosal delivery platform based on the high absorptive capacity of this mucosal surface, the extensive lymphoid tissue, and published preclinical studies demonstrating efficacy of SL vaccination against other pathogens. To this end, we performed preliminary testing of sublingual TB vaccines. Vaccination of mice with SL BCG elicited potent mycobacteria-specific T cell responses which persisted 16 weeks post-immunization. The magnitudes of the T cell responses were similarly induced after sublingual, intranasal, and subcutaneous BCG vaccination. Interestingly, serum mycobacteria-specific antibody responses and systemic recovery of BCG post-vaccination were lower after SL BCG compared with systemic BCG immunization. However, more importantly, SL BCG vaccinated mice were significantly protected against an aerosolized virulent M. tuberculosis challenge (P < 0.0001 compared to unvaccinated mice). Furthermore, this protection was long-lived, persisting for 16 weeks with >1 log CFU reduction compared with naïve challenged mice in both lungs and spleens (P < 0.0001 and P < 0.0028, respectively). These exciting results provide strong support for further studies exploring the mechanisms of protective immunity induced by sublingual BCG vaccination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Failure of the most recent tuberculosis (TB) vaccine trial to boost bacillus Calmette-Guérin-mediated anti-TB immunity despite the induction of Th1-specific central memory cell and effector memory cell responses highlights the importance of identifying optimal T cell targets for protective vaccines. In this study, we describe a novel, Mycobacterium tuberculosis -specific IFN-γ + CD4 + T cell population expressing surface markers characteristic of naive-like memory T cells (T NLM ), which were induced in both human (CD45RA + CCR7 + CD27 + CD95 - ) and murine (CD62L + CD44 - Sca-1 + CD122 - ) systems in response to mycobacteria. In bacillus Calmette-Guérin-vaccinated subjects and those with latent TB infection, T NLM were marked by the production of IFN-γ but not TNF-α and identified by the absence of CD95 expression and increased surface expression CCR7, CD27, the activation markers T-bet, CD69, and the survival marker CD74. Increased tetramer-positive T NLM frequencies were noted in the lung and spleen of ESAT-6 1-20 -specific TCR transgenic mice at 2 wk postinfection with M. tuberculosis and progressively decreased at later time points, a pattern not seen with TNF-α + CD4 + T cells expressing naive cell surface markers. Importantly, adoptive transfer of highly purified T NLM alone, from vaccinated ESAT-6 1-20 -specific TCR transgenic mice, conferred equivalent protection against M. tuberculosis infection in the lungs of Rag -/- mice when compared with total memory populations (central and effector memory cells). Thus, T NLM may represent a memory T cell population that, if optimally targeted, may significantly improve future TB vaccine responses., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Background : Intravesical bacillus Calmette-Guérin (BCG) is the gold standard immunologic agent for treating patients with high-grade non-muscle invasive bladder cancer (NMIBC). Nevertheless, relapse rates remain high and BCG unresponsive NMIBC often requires bladder removal. Preclinical data suggest that priming with percutaneous BCG vaccine could improve response to intravesical BCG. Methods : A single-arm trial (NCT02326168) was performed to study the safety, immunogenicity, and preliminary efficacy of priming. Percutaneous BCG was given 21 days prior to intravesical BCG instillation in patients ( n = 13) with high-risk NMIBC. Immune responses were monitored and compared to a sequentially enrolled cohort of nine control patients receiving only intravesical BCG. The effect of BCG on natural killer (NK) and γδ T cell in vitro cytotoxicity was tested. γδ T cell subsets were determined by T cell receptor gene expression with NanoString. Results : Priming was well tolerated and caused no grade ≥3 adverse events. The 3-month disease-free rate for prime patients was 85% (target goal ≥ 75%). Priming boosted BCG-specific immunity at 3 months and increased the activation status of in vitro expanded circulating NK and γδ T cells and their cytotoxicity against bladder cancer cells through receptor NKG2D. BCG enhanced the cytotoxicity of NK and γδ T cells against K562, RT4, and UM-UC6 but not against T24, UM-UC-3, or UM-UC-14 cells. Infiltrating γδ T cell subsets identified in the bladder includes γ9δ2 and γ8δ2. Conclusions : BCG priming is safe and tolerable. Poor sensitivity to NK and γδ T cell cytotoxicity by some bladder tumors represents a potential BCG-resistance mechanism.