Smoking changes adaptive immunity with persistent effects.

Individuals differ widely in their immune responses, with age, sex and genetic factors having major roles in this inherent variability1-6. However, the variables that drive such differences in cytokine secretion-a crucial component of the host response to immune challenges-remain poorly defined. Here we investigated 136 variables and identified smoking, cytomegalovirus latent infection and body mass index as major contributors to variability in cytokine response, with effects of comparable magnitudes with age, sex and genetics. We find that smoking influences both innate and adaptive immune responses. Notably, its effect on innate responses is quickly lost after smoking cessation and is specifically associated with plasma levels of CEACAM6, whereas its effect on adaptive responses persists long after individuals quit smoking and is associated with epigenetic memory. This is supported by the association of the past smoking effect on cytokine responses with DNA methylation at specific signal trans-activators and regulators of metabolism. Our findings identify three novel variables associated with cytokine secretion variability and reveal roles for smoking in the short- and long-term regulation of immune responses. These results have potential clinical implications for the risk of developing infections, cancers or autoimmune diseases.

Neutralizing antibodies with neurotropic factor treatment maintain neurodevelopmental gene expression upon exposure to human cytomegalovirus.

IMPORTANCE: Human cytomegalovirus (HCMV) infection is the leading cause of non-heritable birth defects worldwide. HCMV readily infects the early progenitor cell population of the developing brain, and we have found that infection leads to significantly downregulated expression of key neurodevelopmental transcripts. Currently, there are no approved therapies to prevent or mitigate the effects of congenital HCMV infection. Therefore, we used human-induced pluripotent stem cell-derived organoids and neural progenitor cells to elucidate the glycoproteins and receptors used in the viral entry process and whether antibody neutralization was sufficient to block viral entry and prevent disruption of neurodevelopmental gene expression. We found that blocking viral entry alone was insufficient to maintain the expression of key neurodevelopmental genes, but neutralization combined with neurotrophic factor treatment provided robust protection. Together, these studies offer novel insight into mechanisms of HCMV infection in neural tissues, which may aid future therapeutic development.

A single, improbable B cell receptor mutation confers potent neutralization against cytomegalovirus.

Cytomegalovirus (CMV) is a leading cause of infant hearing loss and neurodevelopmental delay, but there are no clinically licensed vaccines to prevent infection, in part due to challenges eliciting neutralizing antibodies. One of the most well-studied targets for CMV vaccines is the viral fusogen glycoprotein B (gB), which is required for viral entry into host cells. Within gB, antigenic domain 2 site 1 (AD-2S1) is a target of potently neutralizing antibodies, but gB-based candidate vaccines have yet to elicit robust responses against this region. We mapped the genealogy of B cells encoding potently neutralizing anti-gB AD-2S1 antibodies from their inferred unmutated common ancestor (UCA) and characterized the binding and function of early lineage ancestors. Surprisingly, we found that a single amino acid heavy chain mutation A33N, which was an improbable mutation rarely generated by somatic hypermutation machinery, conferred broad CMV neutralization to the non-neutralizing UCA antibody. Structural studies revealed that this mutation mediated key contacts with the gB AD-2S1 epitope. Collectively, these results provide insight into potently neutralizing gB-directed antibody evolution in a single donor and lay a foundation for using this B cell-lineage directed approach for the design of next-generation CMV vaccines.

Harnessing anti-cytomegalovirus immunity for local immunotherapy against solid tumors.

Tumor infiltration by T cells profoundly affects cancer progression and responses to immunotherapy. However, the tumor immunosuppressive microenvironment can impair the induction, trafficking, and local activity of antitumor T cells. Here, we investigated whether intratumoral injection of virus-derived peptide epitopes could activate preexisting antiviral T cell responses locally and promote antitumor responses or antigen spreading. We focused on a mouse model of cytomegalovirus (CMV), a highly prevalent human infection that induces vigorous and durable T cell responses. Mice persistently infected with murine CMV (MCMV) were challenged with lung (TC-1), colon (MC-38), or melanoma (B16-F10) tumor cells. Intratumoral injection of MCMV-derived T cell epitopes triggered in situ and systemic expansion of their cognate, MCMV-specific CD4+ or CD8+ T cells. The MCMV CD8+ T cell epitopes injected alone provoked arrest of tumor growth and some durable remissions. Intratumoral injection of MCMV CD4+ T cell epitopes with polyinosinic acid:polycytidylic acid (pI:C) preferentially elicited tumor antigen-specific CD8+ T cells, promoted tumor clearance, and conferred long-term protection against tumor rechallenge. Notably, secondary proliferation of MCMV-specific CD8+ T cells correlated with better tumor control. Importantly, intratumoral injection of MCMV-derived CD8+ T cell-peptide epitopes alone or CD4+ T cell-peptide epitopes with pI:C induced potent adaptive and innate immune activation of the tumor microenvironment. Thus, CMV-derived peptide epitopes, delivered intratumorally, act as cytotoxic and immunotherapeutic agents to promote immediate tumor control and long-term antitumor immunity that could be used as a stand-alone therapy. The tumor antigen-agnostic nature of this approach makes it applicable across a broad range of solid tumors regardless of their origin.

Immunoprofiling reveals cell subsets associated with the trajectory of cytomegalovirus reactivation post stem cell transplantation.

Human cytomegalovirus reactivation is a major opportunistic infection after allogeneic haematopoietic stem cell transplantation and has a complex relationship with post-transplant immune reconstitution. Here, we use mass cytometry to define patterns of innate and adaptive immune cell reconstitution at key phases of human cytomegalovirus reactivation in the first 100 days post haematopoietic stem cell transplantation. Human cytomegalovirus reactivation is associated with the development of activated, memory T-cell profiles, with faster effector-memory CD4+ T-cell recovery in patients with low-level versus high-level human cytomegalovirus DNAemia. Mucosal-associated invariant T cell levels at the initial detection of human cytomegalovirus DNAemia are significantly lower in patients who subsequently develop high-level versus low-level human cytomegalovirus reactivation. Our data describe distinct immune signatures that emerged with human cytomegalovirus reactivation after haematopoietic stem cell transplantation, and highlight Mucosal-associated invariant T cell levels at the first detection of reactivation as a marker that may be useful to anticipate the magnitude of human cytomegalovirus DNAemia.

Common T-Cell-Receptor Motifs and Features in Patients with Cytomegalovirus (CMV)-Seronegative End-Stage Renal Disease Receiving a Peptide Vaccination against CMV.

After solid-organ transplantation, reactivation of the cytomegalovirus (CMV) is often observed in seronegative patients and associated with a high risk of disease and mortality. CMV-specific T cells can prevent CMV reactivation. In a phase 1 trial, CMV-seronegative patients with end-stage renal disease listed for kidney transplantation were subjected to CMV phosphoprotein 65 (CMVpp65) peptide vaccination and further investigated for T-cell responses. To this end, CMV-specific CD8+ T cells were characterized by bulk T-cell-receptor (TCR) repertoire sequencing and combined single-cell RNA and TCR sequencing. In patients mounting an immune response to the vaccine, a common SYE(N)E TCR motif known to bind CMVpp65 was detected. CMV-peptide-vaccination-responder patients had TCR features distinct from those of non-responders. In a non-responder patient, a monoclonal inflammatory T-cell response was detected upon CMV reactivation. The identification of vaccine-induced CMV-reactive TCRs motifs might facilitate the development of cellular therapies for patients wait-listed for kidney transplantation.

Human cytomegalovirus protein RL1 degrades the antiviral factor SLFN11 via recruitment of the CRL4 E3 ubiquitin ligase complex.

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of viral immune evasion, targeting intrinsic, innate, and adaptive immunity. We have employed two orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins down-regulated by viral factors expressed during the latest phases of viral infection. This approach revealed that the HIV-1 restriction factor Schlafen-11 (SLFN11) was degraded by the poorly characterized, late-expressed HCMV protein RL1, via recruitment of the Cullin4-RING E3 Ubiquitin Ligase (CRL4) complex. SLFN11 potently restricted HCMV infection, inhibiting the formation and spread of viral plaques. Overall, we show that a restriction factor previously thought only to inhibit RNA viruses additionally restricts HCMV. We define the mechanism of viral antagonism and also describe an important resource for revealing additional molecules of importance in antiviral innate immunity and viral immune evasion.

Neutralizing Antibodies Limit Cell-Associated Spread of Human Cytomegalovirus in Epithelial Cells and Fibroblasts.

Human cytomegalovirus (HCMV) can cause severe clinical disease in immunocompromised individuals, such as allograft recipients and infants infected in utero. Neutralizing activity of antibodies, measured as the ability to prevent the entry of cell-free virus, has been correlated with the reduction in HCMV transmission and the severity of HCMV-associated disease. However, in vivo HCMV amplification may occur mainly via cell-to-cell spread. Thus, quantifying the inhibition of cell-to-cell transmission could be important in the evaluation of therapeutic antibodies and/or humoral responses to infection or immunization. Here, we established a quantitative plaque reduction assay, which allowed for the measurement of the capacity of antibodies to limit HCMV spread in vitro. Using an automated fluorescence spot reader, infection progression was assayed by the expansion of viral plaques during the course of infection with various GFP-expressing viruses. We found that in contrast to non-neutralizing monoclonal antibodies (mAbs), neutralizing mAbs against both glycoprotein B and H (gB and gH) could significantly inhibit viral plaque expansion of different HCMV strains and was equally efficient in fibroblasts as in epithelial cells. In contrast, an anti-pentamer mAb was active only in epithelial cells. Taken together, our data demonstrate that specific anti-HCMV mAbs can significantly limit cell-associated virus spread in vitro.

Dominant Antiviral CD8+ T Cell Responses Empower Prophylactic Antibody-Eliciting Vaccines Against Cytomegalovirus.

Human cytomegalovirus (HCMV) is an ubiquitous herpesvirus that can cause serious morbidity and mortality in immunocompromised or immune-immature individuals. A vaccine that induces immunity to CMV in these target populations is therefore highly needed. Previous attempts to generate efficacious CMV vaccines primarily focused on the induction of humoral immunity by eliciting neutralizing antibodies. Current insights encourage that a protective immune response to HCMV might benefit from the induction of virus-specific T cells. Whether addition of antiviral T cell responses enhances the protection by antibody-eliciting vaccines is however unclear. Here, we assessed this query in mouse CMV (MCMV) infection models by developing synthetic vaccines with humoral immunity potential, and deliberately adding antiviral CD8+ T cells. To induce antibodies against MCMV, we developed a DNA vaccine encoding either full-length, membrane bound glycoprotein B (gB) or a secreted variant lacking the transmembrane and intracellular domain (secreted (s)gB). Intradermal immunization with an increasing dose schedule of sgB and booster immunization provided robust viral-specific IgG responses and viral control. Combined vaccination of the sgB DNA vaccine with synthetic long peptides (SLP)-vaccines encoding MHC class I-restricted CMV epitopes, which elicit exclusively CD8+ T cell responses, significantly enhanced antiviral immunity. Thus, the combination of antibody and CD8+ T cell-eliciting vaccines provides a collaborative improvement of humoral and cellular immunity enabling enhanced protection against CMV.

MCMV-based vaccine vectors expressing full-length viral proteins provide long-term humoral immune protection upon a single-shot vaccination.

Global pandemics caused by influenza or coronaviruses cause severe disruptions to public health and lead to high morbidity and mortality. There remains a medical need for vaccines against these pathogens. CMV (cytomegalovirus) is a ß-herpesvirus that induces uniquely robust immune responses in which remarkably large populations of antigen-specific CD8+ T cells are maintained for a lifetime. Hence, CMV has been proposed and investigated as a novel vaccine vector for expressing antigenic peptides or proteins to elicit protective cellular immune responses against numerous pathogens. We generated two recombinant murine CMV (MCMV) vaccine vectors expressing hemagglutinin (HA) of influenza A virus (MCMVHA) or the spike protein of severe acute respiratory syndrome coronavirus 2 (MCMVS). A single injection of MCMVs expressing either viral protein induced potent neutralizing antibody responses, which strengthened over time. Importantly, MCMVHA-vaccinated mice were protected from illness following challenge with the influenza virus, and we excluded that this protection was due to the effects of memory T cells. Conclusively, we show here that MCMV vectors induce not only long-term cellular immunity but also humoral responses that provide long-term immune protection against clinically relevant respiratory pathogens.

Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system of unknown etiology. We tested the hypothesis that MS is caused by Epstein-Barr virus (EBV) in a cohort comprising more than 10 million young adults on active duty in the US military, 955 of whom were diagnosed with MS during their period of service. Risk of MS increased 32-fold after infection with EBV but was not increased after infection with other viruses, including the similarly transmitted cytomegalovirus. Serum levels of neurofilament light chain, a biomarker of neuroaxonal degeneration, increased only after EBV seroconversion. These findings cannot be explained by any known risk factor for MS and suggest EBV as the leading cause of MS.

Large-scale manufacturing and characterization of CMV-CD19CAR T cells.

BACKGROUND: Adoptive transfer of CD19-specific chimeric antigen receptor (CD19CAR) T cells can induce dramatic disease regression in patients with B cell malignancies. CD19CAR T cell therapy may be limited by insufficient engraftment and persistence, resulting in tumor relapse. We previously demonstrated a proof of principle that cytomegalovirus (CMV)-specific T cells can be isolated and enriched prior to CD19CAR transduction to produce CMV-CD19CAR T cells, and that these CMV-CD19CAR T cells can be expanded in vivo through CMV vaccination, resulting in better tumor control in a murine model. Here we developed a clinical platform for generating CMV-CD19CAR T cells. METHODS: Peripheral blood mononuclear cells (PBMCs) collected from CMV-seropositive healthy donors were stimulated with a good manufacturing practices-grade PepTivator overlapping CMVpp65 peptide pool and enriched for CMV-responsive interferon γ (IFNγ)+T cells using IFNγ Catchmatrix, within the CliniMACS Prodigy Cytokine Capture System (Miltenyi Biotec). Resulting CMV-specific T cells were transduced with a lentiviral vector encoding a second generation CD19R:CD28:ζ/EGFRt CAR and expanded with interleukin 2 (IL-2) and IL-15 for 15 days before characterization. RESULTS: CMV-specific T cells were enriched from 0.8%±0.5 of input PBMC to 76.3%±11.6 in nine full-scale qualification runs (absolute yield of 4.2±3.3×106 IFNγ+T cells from an input of 1×109 PBMCs). Average CD19CAR transduction efficiency of CMV-specific T cells was 27.0%±14.2 in the final products, which underwent rapid expansion, resulting in a total cell dose of 6.2±0.9 × 106 CD19CAR-tranduced T cells with CMV specificity (ie, functionally bispecific). CMV-CD19CAR T cells were polyclonal, expressed memory markers but had low expression of exhaustion markers, responded to both CD19 and CMVpp65 stimulation with rapid proliferation and exhibited antigen-specific effector functions against both CD19-expressing tumors and CMVpp65 antigen. The final products passed release criteria for clinical use. CONCLUSIONS: We demonstrated the feasibility of our large-scale platform for generating CMV-CD19CAR T cells for clinical application. We plan to initiate a clinical trial at City of Hope using CMV-CD19CAR T cells for patients with intermediate/high-grade B cell non-Hodgkin's lymphoma immediately after autologous hematopoietic cell transplantation followed by vaccination with a novel CMV vaccine based on Modified Vaccinia Ankara (Triplex) 28 days and 56 days post-T cell infusion.

Congenital cytomegalovirus infection after maternal primary infection in a patient with systemic lupus erythematosus: A case report and literature review.

BACKGROUND: Management of infectious complications in pregnant women receiving immunosuppressive therapy for systemic lupus erythematosus (SLE) is important. Maternal infection with cytomegalovirus (CMV) often causes congenital CMV infection in the foetus. Thus far, there are only few reports on congenital CMV infection after maternal reactivation in patients with SLE. We report the first case of congenital CMV infection after maternal primary infection in a patient with SLE. CASE PRESENTATION: A 19-year-old Japanese primigravida with SLE received treatment with prednisolone 3 mg/day and azathioprine 75 mg/day at conception. At 7 weeks of gestation, she suddenly developed fever and had decreased white blood cell and platelet counts and elevated aspartate aminotransferase and alanine aminotransferase levels. These clinical findings led to a diagnosis of SLE exacerbation. The prednisolone dose was increased to 15 mg/day, and hydroxychloroquine (200 mg/day) was administered. Consequently, all clinical findings normalised at 12 weeks. At 19 weeks, foetal ultrasound findings revealed oligohydramnios, brain hypoplasia, ventriculomegaly and hyperechogenic bowel. Maternal serological test results indicated increased CMV-specific IgG and IgM levels, low IgG avidity (26%), and positive CMV antigenemia. The foetus was diagnosed with symptomatic congenital CMV infection transmitted from the maternal primary infection. After counselling about the severe prognosis of the foetus, the mother decided to terminate her pregnancy and underwent artificial abortion at 21 weeks. DISCUSSION: The foetus of a mother with SLE who is receiving immunosuppressive therapy may be at increased risk of transmission and aggravation of congenital CMV infection; thus, preventive management and screening for congenital CMV infection during pregnancy are recommended for such patients. Maternal CMV infection shows clinical findings similar to those of SLE exacerbation, and careful differential diagnosis by maternal serological evaluation and foetal ultrasound scans is required.

Cytomegalovirus specific polyfunctional T-cell responses expressing CD107a predict control of CMV infection after liver transplantation.

Cytomegalovirus (CMV) viral load after liver transplantation (LT) is controlled by cell mediated immune responses (CMI). Quantification of CMV-specific T-cells may identify patients who control CMV spontaneously and avoid expensive and potentially toxic antiviral therapies. Prospective post-LT clinical, virological and immunological monitoring was carried out up to 1-year post-LT in a cohort of adult recipients. The CMV-specific T-cell response was characterized using flow cytometry intracellular cytokine staining in 49 LT recipients-R (79.6% R+, 20.4% R-). CMV infection occurred in 24 patients (18 D+/R+ and 6 D+/R-). Only patients with undetectable polyfunctional CMV-specific CD4+ T-cells developed CMV infection. Predictive models showed that polyfunctional CMV-specific CD4+ T-cells pre-existing before LT are protective for CMV reactivation posttransplantation. Quantitation of CD4+ T-cell responses to CMV may be a useful marker for spontaneous control of viral replication to tailor antiviral prophylaxis after LT.

The Direct Influence of Cytomegalovirus Lysate on the Natural Killer Cell Receptor Repertoire.

Natural killer (NK) cells are essential for controlling certain viral infections, including cytomegalovirus (CMV). In particular, the importance of NK cells in the context of CMV infection is underscored by the adaptive capabilities of these cells. Evidence suggests that some viruses can directly interfere with NK cell compartments and their activation and lead to shape-shifting the NK cell receptor repertoire. Still, it remains unknown whether the CMV can interact with NK cells without intermediaries. Here, we examined whether the direct effects of CMV lysate alter phenotypical properties of NK cells. To investigate this issue, NK cells were isolated from the blood of CMV seropositive healthy donors by negative magnetic separation. Isolated NK cells were cultured in the presence of CMV lysate and analyzed for the expression of NKG2A, NKG2C, and CD57 by FACS caliber. The results showed that NKG2C expression is significantly upregulated in the presence of CMV lysate compared to without stimulated group (mean increase, 6.65 %; 95% CI, 0.2582 to 13.02; p=0.043; R square: 0.38). Likewise, results have shown a significant decrease in the frequency of NKG2A+CD57- NK cell subsets (p=0.005; 95% CI, -13.49 to -3.151; R square: 0.5957) in the stimulated group compared to without stimulated ones. According to these results, CMV may drive a direct influence on NK cell receptor repertoire, including the expansion of NK cells expressing NKG2C receptor, which is needed for further studies.

Heterogeneity of human anti-viral immunity shaped by virus, tissue, age, and sex.

The persistence of anti-viral immunity is essential for protection and exhibits profound heterogeneity across individuals. Here, we elucidate the factors that shape maintenance and function of anti-viral T cell immunity in the body by comprehensive profiling of virus-specific T cells across blood, lymphoid organs, and mucosal tissues of organ donors. We use flow cytometry, T cell receptor sequencing, single-cell transcriptomics, and cytokine analysis to profile virus-specific CD8+ T cells recognizing the ubiquitous pathogens influenza and cytomegalovirus. Our results reveal that virus specificity determines overall magnitude, tissue distribution, differentiation, and clonal repertoire of virus-specific T cells. Age and sex influence T cell differentiation and dissemination in tissues, while T cell tissue residence and functionality are highly correlated with the site. Together, our results demonstrate how the covariates of virus, tissue, age, and sex impact the anti-viral immune response, which is important for targeting, monitoring, and predicting immune responses to existing and emerging viruses.

Combined Analysis of Early CD4+ T Cell Counts and CMV Serostatus May Improve CMV Risk Assessment after Allogeneic Hematopoietic Cell Transplantation.

The incidence and severity of viral complications after cellular therapy are highly variable. Recent publications describe relevant interactions between the human Cytomegalovirus (CMV) and host immunity in recipients of allogeneic hematopoietic cell transplantation (HCT). Although immune monitoring is routinely performed in HCT patients, validated cut-off levels correlating with transplant outcomes such as survival or CMV reactivation are mostly limited to day +100, which is later than the median time for CMV reactivation in the absence of medical prophylaxis. To address this gap in early risk assessment, we applied an unsupervised machine learning technique based on clustering of day +30 CD4+ helper T cell count data, and identified relevant cut-off levels within the diverse spectrum of early CD4+ reconstitution. These clusters were stratified for CMV recipient serostatus to identify early risk groups that predict clinical HCT outcome. Indeed, the new risk groups predicted subsequent clinical events such as NRM, OS, and high CMV peak titers better than the most established predictor, i.e., the positive CMV recipient serostatus (R+). More specifically, patients from the R+/low CD4+ subgroup strongly associated with high CMV peak titers and increased 3-year NRM (subdistribution hazard ratio (SHR) 10.1, 95% CI 1.38-73.8, p = 0.023), while patients from the R-/very high CD4+ subgroup showed comparable NRM risks (SHR 9.57, 95% CI 1.12-81.9, p = 0.039) without such an association. In short, our study established novel cut-off levels for early CD4+ T cells via unsupervised learning and supports the integration of host cellular immunity into clinical risk-assessment after HCT in the context of CMV reactivation.