Soluble Immune Factors Research Articles

Immune Biomarkers at Birth Predict Lower Respiratory Tract Infection Risk in a Large Birth Cohort.

Lower respiratory tract infections (LRTIs) remain the leading cause of infant morbidity and mortality worldwide and affect long-term respiratory health. Identifying immunological determinants of LRTI susceptibility may help stratify disease risk and identify therapies. This study aimed to identify neonatal immunological factors predicting LRTI risk in infancy. Cord blood plasma from 191 neonates from the Boston Birth Cohort was analyzed for 28 soluble immune factors. LRTI was defined as bronchiolitis, bronchitis, or pneumonia during the first year of life. Welch's t-test demonstrated significantly higher log10 transformed concentrations of IL-17 and IFNγ in the LRTI group compared to neonates without LRTI in the first year of life (p < 0.05). Risk associations were determined using multivariate survival models. There were 29 infants with LRTIs. High cord blood levels of IFNγ (aHR = 2.35, 95% CI 1.07-5.17), TNF-β (aHR = 2.86, 95% CI 1.27-6.47), MIP-1α (aHR = 2.82, 95% CI 1.22-6.51), and MIP-1β (aHR = 2.34, 95% CI 1.05-5.20) were associated with a higher risk of LRTIs. RANTES was associated with a lower risk (aHR = 0.43, 95% CI 0.19-0.97). Soluble immune factors linked to antiviral immunity (IFNγ) and cytokines mediating inflammatory responses (TNF-β), and cell homing (MIP-1α/b), at birth were associated with an increased risk of LRTIs during infancy.

Insertive vaginal sex is associated with altered penile immunology and enrichment of Gardnerella vaginalis in uncircumcised Ugandan men.

HIV susceptibility is linked to the penile immune milieu (particularly IL-8 levels) and microbiome. The effects of insertive vaginal sex itself on penile immunology and microbiota are not well described. We compared the immune milieu and microbiology of the coronal sulcus (CS) and distal urethra in 47 uncircumcised Ugandan men reporting ever (n=42) or never (n=5) having had vaginal intercourse. Soluble immune factors were assayed by multiplex ELISA, and penile bacteria abundance by 16S rRNA qPCR and sequencing. Co-primary endpoints were penile levels of IL-8 and soluble E-cadherin. Independent of classical STIs, men reporting prior vaginal sex demonstrated elevated IL-8 levels in both the coronal sulcus (1.78vs. 0.81 log10pg/mL, p=.021) and urethra (2.93vs. 2.30 log10pg/mL; p=.003), with a strong inverse relationship between urethral IL-8 levels and the time from last vaginal sex (r=-0.436; p=.004). Vaginal sex was also associated with elevated penile IL-1α/β and soluble E-cadherin (sEcad), a marker of epithelial disruption. Gardnerella vaginalis (Gv) was only present in the penile microbiome of men reporting prior vaginal sex, and urethral Gv absolute abundance was strongly associated with urethral inflammation (r=0.556; p<.001); corynebacteria were enriched in the CS of men reporting no prior vaginal sex and were associated with reduced CS inflammation. Sexual intercourse was associated with sustained changes in penile immunology, potentially mediated through microbial alterations, in particular the urethral abundance of G. vaginalis. Future studies should further characterize the effects of sexual debut on penile bacteria and immunology.

The cellular and immunological dynamics of early and transitional human milk

Human milk is essential for infant nutrition and immunity, providing protection against infections and other immune-mediated diseases during the lactation period and beyond in later childhood. Milk contains a broad range of bioactive factors such as nutrients, hormones, enzymes, immunoglobulins, growth factors, cytokines, and antimicrobial factors, as well as heterogeneous populations of maternal cells. The soluble and cellular components of milk are dynamic over time to meet the needs of the growing infant. In this study, we utilize systems-approaches to define and characterize 62 analytes of the soluble component, including immunoglobulin isotypes, as well as the cellular component of human milk during the first two weeks postpartum from 36 mothers. We identify soluble immune and growth factors that are dynamic over time and could be utilized to classify milk into different phenotypic groups. We identify 24 distinct populations of both epithelial and immune cells by single-cell transcriptome analysis of 128,016 human milk cells. We found that macrophage populations have shifting inflammatory profiles during the first two weeks of lactation. This analysis provides key insights into the soluble and cellular components of human milk and serves as a substantial resource for future studies of human milk.

Immune signatures in variant syndromes of primary biliary cholangitis and autoimmune hepatitis.

Variant syndromes of autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) share diagnostic features of both entities, but their immunological underpinnings remain largely unexplored. We performed blood profiling of 23 soluble immune markers and immunogenetics in a cohort of 88 patients with autoimmune liver diseases (29 typical AIH, 31 typical PBC and 28 with clinically PBC/AIH variant syndromes). The association with demographical, serological and clinical features was analyzed. While T and B cell receptor repertoires were highly skewed in variant syndromes compared to healthy controls, these biases were not sufficiently discriminated within the spectrum of autoimmune liver diseases. High circulating checkpoint molecules sCD25, sLAG-3, sCD86 and sTim-3 discriminated AIH from PBC on top of classical parameters such as transaminases and immunoglobulin levels. In addition, a second cluster of correlated soluble immune factors encompassing essentially TNF, IFNγ, IL12p70, sCTLA-4, sPD-1 and sPD-L1 appeared characteristic of AIH. Cases with complete biochemical responses to treatment generally showed a lower level of dysregulation. Unsupervised hierarchical clustering of classical and variant syndromes identified two pathological immunotypes consisting predominantly of either AIH or PBC cases. Variant syndromes did not form a separate group, but clustered together with either classical AIH or PBC. Clinically, patient with AIH-like variant syndromes were less likely to be able discontinue immunosuppressive treatment. Our analyses suggest that variants of immune mediated liver diseases may represent an immunological spectrum from PBC to AIH-like disease reflected by their pattern of soluble immune checkpoint molecules rather than separate entities.

Is paternal immune activation just as important as maternal immune activation? Time to rethink the bi-parental immune priming of neurodevelopmental model of schizophrenia

The neurodevelopmental origin of schizophrenia is a paradigmatic etiological construct. Parental exposure to environmental adversities and altered parental immune and stress-regulatory pathways affect the neurodevelopment of the fetus and may enhance the risk of schizophrenia in offspring. With respect to the parental contribution to these neurodevelopmental aberrations, most research has been on the maternal contribution. Maternal Immune activation, induced by maternal infections has been identified as a key pathway in the neurodevelopmental model of schizophrenia. Recent understanding points towards the fact that both the maternal and paternal priming offers survival advantages of the offspring to pathogen exposure. However, notwithstanding common epigenetic mechanisms applicable to both sexes, there is a lack of empirical data on the effect of paternal immune activation on neurodevelopmental aberrations and the consequent risk of schizophrenia in offspring.Large birth-cohort studies suggest a comparable impact of maternal and paternal infections on the risk of schizophrenia in offspring. The link between maternal infection and the risk of schizophrenia is mediated by the elements of the immune system through induction of maternal immune activation and subsequent neural and immune developmental aberrations. The contribution of paternal immune activation to schizophrenia risk has not yet been adequately explored. It is rational to assume that paternal infections might also enhance the risk of schizophrenia in offspring through paternal immune activation leading to neuroimmunodevelopmental aberrations. However, the underlying mechanisms whereby paternal infections as well as paternal immune activation might confer the risk of schizophrenia in the offspring seem to be distinct from maternal infection as the modes of transfer of the maternal and paternal factors are different. Maternal immune priming during gestation seems primarily through the direct effect of soluble immune factors, while the paternal immune priming might be mediated predominantly by epigenetic processes or through the effect of modifying factors in sperm. An account of plausible underlying mechanisms linking paternal immune activation and risk of schizophrenia are highlighted in this article. Furthermore, we propose the possibility of bi-parental immune priming in driving the neuroimmunodevelopmental abnormality as a risk mechanism of schizophrenia. It is envisaged that the neuroimmunodevelopmental aberration-associated risk of schizophrenia could be the result of cumulative effects of maternal as well as paternal immune stimulation. Delineation of the underlying maternal and paternal immune priming mechanism(s) will have important implications in resolving dilemmas of schizophrenia etiology as well as prevention.

Combining TNFR2-Expressing Tregs and IL-6 as Superior Diagnostic Biomarkers for High-Grade Serous Ovarian Cancer Masses.

We hypothesised that the inclusion of immunosuppressive and inflammatory biomarkers in HGSOC patients would improve the sensitivity and specificity of the preoperative marker prediction of malignancy in patients with ovarian masses. We tested a panel of 29 soluble immune factors by multiplex bead immunoassay and 16 phenotypic T cell markers by flow cytometry in pre-treatment blood samples from 66 patients undergoing surgery for suspected ovarian cancer or ovarian cancer risk reduction. The potential diagnostic utility of all parameters was explored using Volcano plots, principal component analysis (PCA) and receiver operator characteristic (ROC) analysis. We also assessed the effect of culturing PBMCs from 20 healthy donors in the presence of malignant ascites fluid. The combination of TNFR2+ Tregs and IL-6 in the pre-treatment blood of patients with advanced HGSOC effectively discriminated patients with benign or malignant ovarian masses. In vitro culturing of the PBMCs of healthy donors in malignant ascites promoted an increase in TNFR2-expressing Tregs, which were decreased following blockade with IL-6 or STAT3 activity. Pre-treatment serum IL-6 and peripheral blood TNFR2+ Tregs may be potential clinical biomarkers that can discriminate patients with malignant compared to benign ovarian cancer masses, and the relationship between IL-6 and TNFR2+ Treg is likely to be mediated via the STAT3 signalling pathway.

Distinct systemic immune networks define severe vs. mild COVID-19 in hematologic and solid cancer patients.

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, has impacted health across all sectors of society. A cytokine-release syndrome, combined with an inefficient response of innate immune cells to directly combat the virus, characterizes the severe form of COVID-19. While immune factors involved in the development of severe COVID-19 in the general population are becoming clearer, identification of the immune mechanisms behind severe disease in oncologic patients remains uncertain. Here we evaluated the systemic immune response through the analysis of soluble blood immune factors and anti-SARS-CoV-2 antibodies within the early days of a positive SARS-CoV-2 diagnostic in oncologic patients. Individuals with hematologic malignancies that went on to die from COVID-19 displayed at diagnosis severe leukopenia, low antibody production against SARS-CoV-2 proteins, and elevated production of innate immune cell recruitment and activation factors. These patients also displayed correlation networks in which IL-2, IL-13, TNF-alpha, IFN-gamma, and FGF2 were the focal points. Hematologic cancer patients that showed highly networked and coordinated anti-SARS-CoV-2 antibody production, with central importance of IL-4, IL-5, IL-12A, IL-15, and IL-17A, presented only mild COVID-19. Conversely, solid tumor patients that had elevated levels of inflammatory cytokines IL-6, CXCL8, and lost the coordinate production of anti-virus antibodies developed severe COVID-19 and died. Patients that displayed positive correlation networks between anti-virus antibodies, and a regulatory axis involving IL-10 and inflammatory cytokines recovered from the disease. We also provided evidence that CXCL8 is a strong predictor of death for oncologic patients and could be an indicator of poor prognosis within days of the positive diagnostic of SARS-CoV-2 infection. Our findings defined distinct systemic immune profiles associated with COVID-19 clinical outcome of patients with cancer and COVID-19. These systemic immune networks shed light on potential immune mechanisms involved in disease outcome, as well as identify potential clinically useful biomarkers.

Treatment Success Following Standard Antibiotic Treatment for Bacterial Vaginosis Is Not Associated With Pretreatment Genital Immune or Microbial Parameters.

Bacterial vaginosis (BV) is a proinflammatory genital condition associated with adverse reproductive health outcomes, including increased HIV incidence. However, BV recurrence rates are high after standard antibiotic treatment. While the composition of the vaginal microbiota before BV treatment may be linked to BV recurrence, it is unclear whether the preceding genital immune milieu is predictive of treatment success. Here we assessed whether baseline vaginal soluble immune factors or the composition of the vaginal microbiota predicted treatment success 1 month after metronidazole treatment in 2 separate cohorts of women with BV, 1 in the United States and 1 in Kenya; samples within 48 hours of BV treatment were also available for the US cohort. Neither soluble immune factors nor the composition of the vaginal microbiota before BV treatment was associated with treatment response in either cohort. In the US cohort, although the absolute abundances of key vaginal bacterial taxa pretreatment were not associated with treatment response, participants with sustained BV clearance had a more pronounced reduction in the absolute abundance of Gardnerella vaginalis immediately after treatment. Pretreatment immune and microbial parameters were not predictive of BV treatment success in these clinical cohorts.

Elevated inflammatory fecal immune factors in men who have sex with men with HIV associate with microbiome composition and gut barrier function.

People living with HIV infection (PLWH) exhibit elevated levels of gastrointestinal inflammation. Potential causes of this inflammation include HIV infection and associated immune dysfunction, sexual behaviors among men who have sex with men (MSM) and gut microbiome composition. To better understand the etiology of gastrointestinal inflammation we examined levels of 28 fecal soluble immune factors (sIFs) and the fecal microbiome in well-defined cohorts of HIV seronegative MSM (MSM-SN), MSM with untreated HIV infection (MSM-HIV) and MSM with HIV on anti-retroviral treatment (MSMART). Additionally, fecal solutes from these participants were used to stimulate T-84 colonic epithelial cells to assess barrier function. Both MSM cohorts with HIV had elevated levels of fecal calprotectin, a clinically relevant marker of GI inflammation, and nine inflammatory fecal sIFs (GM-CSF, ICAM-1, IL-1β, IL-12/23, IL-15, IL-16, TNF-β, VCAM-1, and VEGF). Interestingly, four sIFs (GM-CSF, ICAM-1, IL-7 and IL-12/23) were significantly elevated in MSM-SN compared to seronegative male non-MSM. Conversely, IL-22 and IL-13, cytokines beneficial to gut health, were decreased in all MSM with HIV and MSM-SN respectively. Importantly, all of these sIFs significantly correlated with calprotectin, suggesting they play a role in GI inflammation. Principal coordinate analysis revealed clustering of fecal sIFs by MSM status and significant associations with microbiome composition. Additionally, fecal solutes from participants in the MSM-HIV cohort significantly decreased colonic transcellular fluid transport in vitro, compared to non-MSM-SN, and this decrease associated with overall sIF composition and increased concentrations of eight inflammatory sIFs in participants with HIV. Lastly, elevated levels of plasma, sCD14 and sCD163, directly correlated with decreased transcellular transport and microbiome composition respectively, indicating that sIFs and the gut microbiome are associated with, and potentially contribute to, bacterial translocation. Taken together, these data demonstrate that inflammatory sIFs are elevated in MSM, regardless of HIV infection status, and are associated with the gut microbiome and intestinal barrier function.

Unravelling soluble immune checkpoints in chronic lymphocytic leukemia: Physiological immunomodulators or immune dysfunction.

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape.

Salmonella enterica Infections Are Disrupted by Two Small Molecules That Accumulate within Phagosomes and Differentially Damage Bacterial Inner Membranes.

ABSTRACTGram-negative bacteria have a robust cell envelope that excludes or expels many antimicrobial agents. However, during infection, host soluble innate immune factors permeabilize the bacterial outer membrane. We identified two small molecules that exploit outer membrane damage to access the bacterial cell. In standard microbiological media, neither compound inhibited bacterial growth nor permeabilized bacterial outer membranes. In contrast, at micromolar concentrations, JAV1 and JAV2 enabled the killing of an intracellular human pathogen, Salmonella enterica serovar Typhimurium. S. Typhimurium is a Gram-negative bacterium that resides within phagosomes of cells from the monocyte lineage. Under broth conditions that destabilized the lipopolysaccharide layer, JAV2 permeabilized the bacterial inner membrane and was rapidly bactericidal. In contrast, JAV1 activity was more subtle: JAV1 increased membrane fluidity, altered reduction potential, and required more time than JAV2 to disrupt the inner membrane barrier and kill bacteria. Both compounds interacted with glycerophospholipids from Escherichia coli total lipid extract-based liposomes. JAV1 preferentially interacted with cardiolipin and partially relied on cardiolipin production for activity, whereas JAV2 generally interacted with lipids and had modest affinity for phosphatidylglycerol. In mammalian cells, neither compound significantly altered mitochondrial membrane potential at concentrations that killed S. Typhimurium. Instead, JAV1 and JAV2 became trapped within acidic compartments, including macrophage phagosomes. Both compounds improved survival of S. Typhimurium-infected Galleria mellonella larvae. Together, these data demonstrate that JAV1 and JAV2 disrupt bacterial inner membranes by distinct mechanisms and highlight how small, lipophilic, amine-substituted molecules can exploit host soluble innate immunity to facilitate the killing of intravesicular pathogens.

Abstract 5590: Inhibition of the autophagy protein Vps34 induces tumor cell secretion of proinflammatory chemokines

Abstract Autophagy is the cellular process by which cytoplasmic contents are degraded and recycled through a lysosomal pathway. In cancer, autophagy can contribute to both tumor promotion and tumor suppression, and emerging evidence supports a role in resistance to immunotherapy. We investigated whether inhibition of autophagy with a small molecule inhibitor of the class III phosphoinositide 3-kinase Vps34 could stimulate anti-tumor immune responses and potentiate the efficacy of immune checkpoint blockade (ICB). Treatment with the Vps34 inhibitor (Vps34i) inhibited autophagy and was weakly cytotoxic to B16F10, MC38, EMT6 and CT26 mouse syngeneic tumor cell lines. In certain cancers, autophagy has been linked to lysosomal degradation of MHC I proteins, thereby downregulating expression and contributing to immune evasion. Notably, we observed minimal upregulation of MHC I surface expression on a subset of mouse and human cell lines. We further explored whether autophagy inhibition would result in release of any soluble immune stimulatory factors from mouse tumor cell lines. Inhibition of Vps34 markedly increased the secretion of several proinflammatory chemokines, including CCL5 and CXCL10. Increased chemokine secretion was also observed in a panel of human cancer cell lines, including NCI-H2009 lung cancer and PC-3 prostate cancer, confirming that the response is observed in human cancer cell lines derived from different tissue origins. As the chemokines CCL5 and CXCL10 are critical to the development of robust anti-tumor immunity, we next tested whether Vps34i treatment could potentiate the anti-tumor efficacy of ICB. We explored the activity of Vps34i as a single agent and in combination with anti-PD-1 antibody in three different syngeneic tumor models, including EMT6, CT26 and MC38. No single agent activity was observed in these models, however, combination of anti-PD-1 and Vps34i significantly inhibited the growth of CT26 tumors compared to control. Anti-tumor efficacy of combination treatment was not significantly different compared to single agents, and no complete tumor regressions were observed. No significant increases in CCL5 and CXCL10 were observed in the serum of combination treated mice, potentially explaining the lack of robust efficacy. Our results suggest that inhibition of autophagy promotes the secretion of proinflammatory cytokines from mouse and human cancer cells in vitro. As the autophagy pathway exhibits cross talk with other major cell signaling pathways, the tumor contexture may be a critical determinant of the role of autophagy in anti-tumor immune responses. Future experiments will explore which tumor contexts are sensitive to combination of autophagy inhibition and ICB. Citation Format: Marie Bernardo, Yu-an Zhang, Martin Graf, Jane Cheng, Fangxian Sun, Virna Cortez-Retamozo, Sukhvinder Sidhu, Eladio Marquez, Donald Jackson, Jack Pollard, Timothy R. Wagenaar, Donald Shaffer. Inhibition of the autophagy protein Vps34 induces tumor cell secretion of proinflammatory chemokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5590.

Abstract CT247: A phase I/II trial investigating safety and efficacy of autologous TAC T cells targeting HER2 in relapsed or refractory solid tumors (TACTIC-2)

Abstract Background: Despite recent therapeutic advances for patients (pts) with breast, colorectal and gastroesophageal cancers with HER2 overexpression, there is still a significant unmet medical need for better treatment options for HER2-positive solid tumors, especially those with low or intermediate HER2 expression (1+ and 2+ by immunohistochemistry (IHC)). T Cell Antigen-Coupler (TAC) technology is a novel way to genetically modify T cells and to redirect these T cells to target cancer antigens and to activate T cells naturally by co-opting the natural T cell Receptor (TCR). TAC01-HER2 is an autologous T-cell product comprising T cells expressing the HER2 TAC, a chimeric receptor that is genetically engineered into T cells via lentiviral transduction to furnish T cells with two main functions: redirection to and specific recognition of HER2-positive cells, and T cell activation via the endogenous TCR. In vivo models, TAC T-cells can infiltrate tumors and persist for extended period of time, leading to robust and persistent anti-tumor efficacy, with a favorable safety profile. The safety and efficacy of TAC01-HER2 is investigated in the ongoing (TACTIC-2) first-in-human phase I-II study (NCT04727151). Methods: Eligible pts are ≥ 18 years with HER2-positive solid tumors (1+, 2+ or 3+ by IHC, regardless of amplification status) who progressed after at least two lines of systemic therapy. Upon enrollment, pts undergo leukapheresis to obtain T cells for TAC01-HER2 manufacturing. Bridging therapy can be administered prior to lymphodepleting chemotherapy (LDC). TAC01-HER2 is a single infusion after LDC with a starting dose of 1-3 x 105 cells/kg and escalating based on the keyboard statistical dose escalation method. Data safety monitoring committee meetings will be held after each dose level. Once the recommended phase 2 dose (RP2D) is identified, an additional 3-6 pts will be treated at that dose level. Current planned phase II expansion cohorts include HER2 3+ breast cancer (N=20), HER2 3+ other solid tumors (N=20), and HER2 2+ breast and other solid tumors (N=10). The primary objective for the trial is to determine the safety and tolerability of TAC01-HER2 with primary endpoints of incidence of dose limiting toxicities (DLTs) and type, frequency, and severity of adverse events (AEs). Secondary endpoints include the RP2D, overall response rate, duration of response, overall survival and pharmacokinetics. Exploratory endpoints include assessment of potential biomarkers of response or resistance and characterization of soluble immune factors and relationship to cytokine release syndrome (CRS), neurotoxicity and TAC T cell engraftment. Tumor response assessments are performed at 4 weeks, then at months 3, 6, 9, 12, 18 and 24. After study completion, subjects are followed for survival and long-term safety for up to 15 years. The trial opened in January 2021 and it is actively enrolling pts. Citation Format: Ecaterina E. Dumbrava, Daniel Olson, Benjamin L. Schlechter, Sam Saibil, Donna Rill, Andreas Bader, Deyaa Adib, Michael Bishop. A phase I/II trial investigating safety and efficacy of autologous TAC T cells targeting HER2 in relapsed or refractory solid tumors (TACTIC-2) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT247.

Sustained effect of LACTIN-V (Lactobacillus crispatus CTV-05) on genital immunology following standard bacterial vaginosis treatment: results from a randomised, placebo-controlled trial

SummaryBackgroundBacterial vaginosis might increase HIV risk by eliciting genital inflammation and epithelial barrier disruption, whereas vaginal Lactobacillus crispatus is associated with immune quiescence and HIV protection. We investigated the effect of a live biotherapeutic containing L crispatus CTV-05 (LACTIN-V) on genital immunology and key vaginal bacteria.MethodsThis substudy included women aged 18–45 years who participated in the randomised, placebo-controlled, phase 2b trial of LACTIN-V to reduce bacterial vaginosis recurrence, conducted at four universities and hospitals in the USA. Women with negative results for sexually transmitted infection, pregnancy, and urinary tract infection were provided a 5-day course of vaginal metronidazole 0·75% gel. Those who met at least three of four clinical Amsel criteria for bacterial vaginosis and had a Nugent score of 4–10 from Gram staining were eligible. Participants in the LACTIN-V trial were randomly assigned (2:1) to receive either LACTIN-V or placebo, applied vaginally once per day for 5 days during the first week and then twice per week for 10 more weeks. Follow-up visits occurred 4, 8, 12, and 24 weeks after enrolment. Soluble immune factors and the absolute abundance of bacterial taxa were assayed by mutliplex ELISA and quantitative PCR. The primary outcomes were vaginal levels of IL-1α and soluble E-cadherin at 24 weeks (ie, 13 weeks after treatment cessation).FindingsBetween Feb 21, 2020 and March 18, 2021, we characterised genital immune parameters and the vaginal microbiota in a subset of 66 highly adherent participants who were randomly selected, with no exclusion criteria, from those who had attended all study follow-up visits (n=166) in the larger LACTIN-V clinical trial (n=288). 32 (48%) participants received LACTIN-V and 34 (52%) received placebo. LACTIN-V treatment was significantly associated with lower concentrations of the proinflammatory cytokine IL-1α (β coefficient 0·310, SE 0·149; p=0·042) and soluble E-cadherin (0·429, 0·199; p=0·035), a biomarker of epithelial barrier disruption.InterpretationVaginal administration of LACTIN-V following standard bacterial vaginosis therapy resulted in a sustained reduction in genital inflammation and a biomarker of epithelial integrity. The potential of LACTIN-V to reduce HIV susceptibility merits further investigation.FundingCanadian Institutes of Health Research and the National Institutes of Health National Institute of Allergy and Infectious Diseases.

Immune milieu and microbiome of the distal urethra in Ugandan men: impact of penile circumcision and implications for HIV susceptibility

BackgroundCoronal sulcus (CS) anaerobe abundance and IL-8 levels are linked to HIV acquisition, and are dramatically reduced after penile circumcision (PC). The distal urethra may be the site of some HIV acquisition before PC, and presumably most acquisition post PC. We describe the immune milieu and microbiome of the distal urethra in uncircumcised Ugandan men, and define the impact of PC. Participants consisted of HIV-negative, genital symptom-free adult Ugandan men undergoing PC (n = 51). Urethral and coronal sulcus swabs were collected at baseline and at 6- and 12-months post-PC. Soluble immune factors were quantified by multiplex ELISA, and bacterial abundance assessed by 16S rRNA qPCR and sequencing.ResultsAt baseline, the urethra was enriched compared to the CS for most cytokines (including IL-8 and MIP-1β) and soluble E-cadherin (sE-cadherin, an epithelial disruption marker), although CS levels of IL-1α and IL-1β were higher. Baseline total bacterial abundance was ≥ 20-fold higher in the CS than the urethra (median 27,100 vs. 1200 gene copies/swab, p = 0.001), and anaerobes comprised 58% of CS bacteria vs. 42% of urethral bacteria. PC did not alter urethral IL-8 (median 806 at baseline vs. 1130 pg/ml at 12 months; p = 0.062) and urethral sE-cadherin increased (113,223 vs. 158,385 pg/ml, p = 0.009), despite five- and sevenfold drops in total bacterial and anaerobe abundance after PC, respectively. However, PC dramatically reduced CS levels of sE-cadherin (15,843 vs. 837 pg/ml, p < 0.001) and most cytokines (IL-8; 34 vs. 3 pg/ml, p < 0.001), while reducing total bacterial and anaerobe abundance by 13-fold and 60-fold, respectively (both P ≤ 0.004).ConclusionsThe urethra is immunologically rich with characteristics of an HIV-susceptible tissue site. However, PC had no impact on urethral immunology and may have reduced epithelial integrity, despite modest reductions in total bacteria and anaerobes, suggesting that HIV protection from PC is not mediated via immune or microbiome alterations in the urethra.6PgEasmsZ9wG1E4iwYa-kCVideo abstract

Confounders in Identification and Analysis of Inflammatory Biomarkers in Cardiovascular Diseases.

Proinflammatory biomarkers have been increasingly used in epidemiologic and intervention studies over the past decades to evaluate and identify an association of systemic inflammation with cardiovascular diseases. Although there is a strong correlation between the elevated level of inflammatory biomarkers and the pathology of various cardiovascular diseases, the mechanisms of the underlying cause are unclear. Identification of pro-inflammatory biomarkers such as cytokines, chemokines, acute phase proteins, and other soluble immune factors can help in the early diagnosis of disease. The presence of certain confounding factors such as variations in age, sex, socio-economic status, body mass index, medication and other substance use, and medical illness, as well as inconsistencies in methodological practices such as sample collection, assaying, and data cleaning and transformation, may contribute to variations in results. The purpose of the review is to identify and summarize the effect of demographic factors, epidemiological factors, medication use, and analytical and pre-analytical factors with a panel of inflammatory biomarkers CRP, IL-1b, IL-6, TNFa, and the soluble TNF receptors on the concentration of these inflammatory biomarkers in serum.

Recurrent bacterial vaginosis following metronidazole treatment is associated with microbiota richness at diagnosis

Bacterial vaginosis-a condition defined by a shift from Lactobacillus dominance to a polymicrobial, anaerobic bacterial community-increases the risk of acquiring sexually transmitted infections and other complications of the female reproductive tract. Antibiotic treatment frequently fails to return the microbiome to an optimal Lactobacillus-dominated state. No criteria currently exist to identify the patients likely to experience treatment failure. We sought to identify the pretreatment community signatures associated with treatment failure through 16S ribosomal RNA gene analysis. Twenty-eight women who were enrolled in an oral metronidazole treatment trial of bacterial vaginosis were studied. Cervicovaginal lavage samples were collected before metronidazole treatment and at 7 and 30 days posttreatment. Cervicovaginal lavage DNA was amplified and sequenced using a paired-end, V4 region 2×150 MiSeq run. Of the 28 women, 25% failed to clear bacterial vaginosis; 35.7% demonstrated a transient clearance, shifting to community-type 2 (Lactobacillus iners dominant) at visit 2 only; 7.1% demonstrated a delayed clearance, reaching community-type 2 at the final visit only; and 32.1% of patients experienced sustained bacterial vaginosis clearance. Examination of the community composition and structure demonstrated that both the richness and the evenness were significantly lower for the women who experienced sustained clearance, whereas the women who failed to clear bacterial vaginosis possessed the highest median levels of richness, evenness, and diversity pretreatment. Soluble immune factors in the lower reproductive tract improved significantly following a shift from community-type 4 to a Lactobacillus-dominant microbiome, with the samples categorized as community-type 2 possessing significantly higher levels of secretory leukocyte protease inhibitor, growth-regulated alpha protein, and macrophage inflammatory protein-3 and significantly lower levels of intercellular adhesion molecule-1. Although the shifts to Lactobacillus dominance improved the markers of mucosal tissue health, these gains were only temporary among the women who experienced recurrence. Assemblies of highly diverse microbiota are associated with the enhanced resilience of bacterial vaginosis to standard metronidazole treatment. These communities may be foundational to treatment resistance or simply an indication of a well-established community made possible by canonical biofilm-forming taxa. Future studies must target the transcriptional activity of these communities under the pressure of antibiotic treatment to resolve the mechanisms of their resistance.

Low-Level Anorectal HIV Shedding despite Effective Antiretroviral Therapy Is Not Driven by Mucosal Inflammation.

Although antiretroviral treatment (ART) suppresses HIV RNA in blood and prevents transmission, low-level anorectal HIV RNA shedding persists in some ART-treated men who have sex with men. We collected anorectal biopsies and swabs from 55 men who have sex with men on effective ART, hypothesizing that anorectal shedding would be linked to microbiota-driven mucosal T cell activation. Lymphocytes were assessed by flow cytometry, soluble immune factors by multiplex immunoassay, neutrophils and epithelial integrity by immunofluorescence microscopy, and the anorectal microbiome by quantitative PCR and 16S rRNA gene sequencing. Unexpectedly, we found no evidence that anorectal HIV shedding was associated with the parameters of mucosal inflammation, including T cell activation, inflammatory cytokines, the density of neutrophils, or epithelial integrity. Moreover, the anorectal bacterial load was actually lower in the shedding group, with no major differences in bacterial composition. Instead, the strongest mucosal immune correlates of HIV shedding were an increase in central memory cell frequency and Ki67 expression as well as higher concentrations of the cytokine IL-7 in anorectal secretions. Anorectal HIV RNA shedding during effective ART was not driven by local inflammation; the associations seen with local homeostatic T cell proliferation will require further confirmation.

The Power of First Impressions: Can Influenza Imprinting during Infancy Inform Vaccine Design?

Influenza virus infection causes severe respiratory illness in people worldwide, disproportionately affecting infants. The immature respiratory tract coupled with the developing immune system, and lack of previous exposure to the virus is thought to synergistically play a role in the increased disease severity in younger age groups. No influenza vaccines are available for those under six months, although maternal influenza immunization is recommended. In children aged six months to two years, vaccine immunogenicity is dampened compared to older children and adults. Unlike older children and adults, the infant immune system has fewer antigen-presenting cells and soluble immune factors. Paradoxically, we know that a person’s first infection with the influenza virus during infancy or childhood leads to the establishment of life-long immunity toward that particular virus strain. This is called influenza imprinting. We contend that by understanding the influenza imprinting event in the context of the infant immune system, we will be able to design more effective influenza vaccines for both infants and adults. Working through the lens of imprinting, using infant influenza animal models such as mice and ferrets which have proven useful for infant immunity studies, we will gain a better understanding of imprinting and its implications regarding vaccine design. This review examines literature regarding infant immune and respiratory development, current vaccine strategies, and highlights the importance of research into the imprinting event in infant animal models to develop more effective and protective vaccines for all including young children.

Type I IFN induces vascular permeability delivering iron binding proteins to the site of Cryptococcosis gattii infection resulting in local nutritional immunity and contained infection

Abstract While Cryptococcus neoformans primarily infect AIDS patients, Cryptococcus gattii (Cg) causes deadly mycosis in mostly non-HIV patients even in Cg endemic areas with high rates of AIDS. Previous work found that type I IFN (t1IFN), which is induced by HIV infection, protects mice from Cg. Thus, we sought to examine the mechanisms mediating t1IFN protection from Cg. Previous data ruled out cellular immunity, so we examined soluble immune factors. We found that induction of t1IFN by stabilized poly(I:C) (pICLC) induced the expression of serum proteins into the lung airspace fluids. PICLC induced the leak of intravenously injected FITC-dextran into lung airspace fluids suggesting that serum proteins reach the lung airspace via t1IFN-induced vascular permeability. The pICLC-mediated expression of serum proteins into the lung airspace was dependent on t1IFN but not IL-6, IL-1 or TNF-α indicating that this effect is mediated by t1IFN and not by other cytokines characterized to affect vascular permeability. Transferrin and ferritin were among the leaked serum proteins found in the lung airspace and were directly inhibitory to cryptococcus in vitro so we hypothesized that the presence of these proteins in the lung airspace may result in local iron deficiency. Iron supplementation reversed pICLC-protection while iron chelation mediated similar protection as pICLC. Together these data suggest that t1IFN induces vascular permeability in the lung which delivers iron binding proteins to the Cg niche. These host proteins restrict iron to Cg and limit infection. In addition to explaining the pICLC-induced protection from Cg, these studies illustrate a novel connection between t1IFN and vascular permeability that may be significant to other systems.