QuantiFERON®-CMV assay for the assessment of cytomegalovirus cell-mediated immunity.

Cytomegalovirus (CMV) infection has historically been a major complication among immunocompromised patients, such as solid-organ and stem-cell transplant recipients and patients with advanced HIV infection. While the introduction of antiretroviral therapy has almost eradicated CMV infection in HIV-infected patients, CMV disease remains a significant problem in transplant recipients once antiviral prophylaxis is discontinued. QuantiFERON(®)-CMV allows the assessment of cellular immunity against CMV by detecting the production of IFN-γ following in vitro stimulation with CMV antigens. Preliminary studies have shown a correlation between a lack of detectable cell-mediated immunity measured by the QuantiFERON-CMV assay and a higher incidence of CMV infection and disease in immunocompromised patients. Measurement of cell-mediated immunity against CMV appears to be a promising strategy to identify patients at highest risk for the development of CMV disease and, therefore, to individualize preventive strategies for CMV in transplant recipients.

Cell-mediated immunity to predict cytomegalovirus disease in high-risk solid organ transplant recipients.

Late-onset cytomegalovirus (CMV) disease commonly occurs after discontinuation of antiviral prophylaxis. We determined the utility of testing CD8+ T-cell response against CMV as a predictor of late-onset CMV disease after a standard course of antiviral prophylaxis. Transplant patients at high-risk for CMV disease were enrolled. CD8+ T-cell-mediated immunity (CMI) was tested using the QuantiFERON-CMV assay at baseline, 1, 2 and 3 months posttransplant by measurement of interferon-gamma response to whole blood stimulation with a 21-peptide pool. The primary outcome was the ability of CMI testing to predict CMV disease in the first 6 months posttransplant. There were 108 evaluable patients (D+/R+ n = 39; D-/R+ n = 34; D+/R- n = 35) of whom 18 (16.7%) developed symptomatic CMV disease. At the end of prophylaxis, CMI was detectable in 38/108 (35.2%) patients (cutoff 0.1 IU/mL interferon-gamma). CMV disease occurred in 2/38 (5.3%) patients with a detectable interferon-gamma response versus 16/70 (22.9%) patients with a negative response; p = 0.038. In the subgroup of D+/R- patients, CMV disease occurred in 1/10 (10.0%) patients with a detectable interferon-gamma response (cutoff 0.1 IU/mL) versus 10/25 (40.0%) patients with a negative CMI, p = 0.12. Monitoring of CMI may be useful for predicting late-onset CMV disease.

Assessment of cytomegalovirus-specific cell-mediated immunity for the prediction of cytomegalovirus disease in high-risk solid-organ transplant recipients: a multicenter cohort study.

BACKGROUND: Cytomegalovirus (CMV) disease remains an important problem in solid-organ transplant recipients, with the greatest risk among donor CMV-seropositive, recipient-seronegative (D(+)/R(-)) patients. CMV-specific cell-mediated immunity may be able to predict which patients will develop CMV disease. METHODS: We prospectively included D(+)/R(-) patients who received antiviral prophylaxis. We used the Quantiferon-CMV assay to measure interferon-γ levels following in vitro stimulation with CMV antigens. The test was performed at the end of prophylaxis and 1 and 2 months later. The primary outcome was the incidence of CMV disease at 12 months after transplant. We calculated positive and negative predictive values of the assay for protection from CMV disease. RESULTS: Overall, 28 of 127 (22%) patients developed CMV disease. Of 124 evaluable patients, 31 (25%) had a positive result, 81 (65.3%) had a negative result, and 12 (9.7%) had an indeterminate result (negative mitogen and CMV antigen) with the Quantiferon-CMV assay. At 12 months, patients with a positive result had a subsequent lower incidence of CMV disease than patients with a negative and an indeterminate result (6.4% vs 22.2% vs 58.3%, respectively; P < .001). Positive and negative predictive values of the assay for protection from CMV disease were 0.90 (95% confidence interval [CI], .74-.98) and 0.27 (95% CI, .18-.37), respectively. CONCLUSIONS: This assay may be useful to predict if patients are at low, intermediate, or high risk for the development of subsequent CMV disease after prophylaxis. CLINICAL TRIALS REGISTRATION: NCT00817908.

Synthetic long peptide-based vaccine formulations for induction of cell mediated immunity: A comparative study of cationic liposomes and PLGA nanoparticles.

Nanoparticulate formulations for synthetic long peptide (SLP)-cancer vaccines as alternative to clinically used Montanide ISA 51- and squalene-based emulsions are investigated in this study. SLPs were loaded into TLR ligand-adjuvanted cationic liposomes and PLGA nanoparticles (NPs) to potentially induce cell-mediated immune responses. The liposomal and PLGA NP formulations were successfully loaded with up to four different compounds and were able to enhance antigen uptake by dendritic cells (DCs) and subsequent activation of T cells in vitro. Subcutaneous vaccination of mice with the different formulations showed that the SLP-loaded cationic liposomes were the most efficient for the induction of functional antigen-T cells in vivo, followed by PLGA NPs which were as potent as or even more than the Montanide and squalene emulsions. Moreover, after transfer of antigen-specific target cells in immunized mice, liposomes induced the highest in vivo killing capacity. These findings, considering also the inadequate safety profile of the currently clinically used adjuvant Montanide ISA-51, make these two particulate, biodegradable delivery systems promising candidates as delivery platforms for SLP-based immunotherapy of cancer.

Counteraction of HLA-C-mediated immune control of HIV-1 by Nef.

A host genetic variant (-35C/T) correlates with increased human leukocyte antigen C (HLA-C) expression and improved control of HIV-1. HLA-C-mediated immunity may be particularly protective because HIV-1 is unable to remove HLA-C from the cell surface, whereas it can avoid HLA-A- and HLA-B-mediated immunity by Nef-mediated down-modulation. However, some individuals with the protective -35CC genotype exhibit high viral loads. Here, we investigated whether the ability of HIV-1 to replicate efficiently in the "protective" high-HLA-C-expression host environment correlates with specific functional properties of Nef. We found that high set point viral loads (sVLs) were not associated with the emergence of Nef variants that had acquired the ability to down-modulate HLA-C or were more effective in removing HLA-A and HLA-B from the cell surface. However, in individuals with the protective -35CC genotype we found a significant association between sVLs and the efficiency of Nef-mediated enhancement of virion infectivity and modulation of CD4, CD28, and the major histocompatibility complex class II (MHC-II)-associated invariant chain (Ii), while this was not observed in subjects with the -35TT genotype. Since the latter Nef functions all influence the stimulation of CD4(+) T helper cells by antigen-presenting cells, they may cooperate to affect both the activation status of infected T cells and the generation of an antiviral cytotoxic T-lymphocyte (CTL) response. In comparison, different levels of viremia in individuals with the common -35TT genotype were not associated with differences in Nef function but with differences in HLA-C mRNA expression levels. Thus, while high HLA-C expression may generally facilitate control of HIV-1, Nef may counteract HLA-C-mediated immune control in some individuals indirectly, by manipulating T-cell function and MHC-II antigen presentation.

Thymoma, immunodeficiency, and herpes simplex virus infections.

Hypogammaglobulinemia develops in 3 to 6% of patients with thymoma and this association is commonly referred to as thymoma with immunodeficiency (formerly Good syndrome). Recurrent infections with encapsulated bacteria and opportunistic infections associated with disorders of both humoral and cell mediated immunity frequently occur in this rare primary, adult-onset immunodeficiency. We report a case of thymoma with immunodeficiency complicated by disseminated herpes simplex virus (HSV) infection and review five additional cases of HSV-related infections reported since 1966 in patients presenting with thymoma with immunodeficiency. Patients presented with epiglottitis, keratitis, recurrent genital herpes, ulcerative dermatitis, and acute hepatitis. Four of the six cases had a fatal outcome, two of which were directly attributable to HSV infection. Since the risk of invasive opportunistic infections is high and the presentation atypical, lymphocyte count and total serum immunoglobulin should be measured regularly in all patients presenting with thymoma with immunodeficiency.

Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant.

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene-5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile.

Attenuated poxviruses expressing a synthetic HIV protein stimulate HLA-A2-restricted cytotoxic T-cell responses.

Efficient HIV vaccines have to trigger cell-mediated immunity directed against various viral antigens. However little is known about the breadth of the response induced by vaccines carrying multiple proteins. Here, we report on the immunogenicity of a construct harbouring a fusion of the HIV-1 IIIB gag, pol and nef genes (gpn) designed for optimal safety and equimolar expression of the HIV proteins. The attenuated poxviruses, MVA and NYVAC, harbouring the gpn construct, induced potent immune responses in conventional mice characterised by stimulation of Gpn-specific IFN-gamma-producing cells and cytotoxic T cells. In HLA-A2 transgenic mice, recombinant MVA elicited cytotoxic responses against epitopes recognised in most HLA-A2+ HIV-1-infected individuals. We also found that the MVA vaccine triggered the in vitro expansion of peripheral blood cells isolated from a HIV-1-seropositive patient and with similar specificity as found in immunised HLA-A2 transgenic mice. In conclusion, the synthetic HIV polyantigen Gpn delivered by MVA is immunogenic, efficiently processed and presented by human MHC class I molecules.

SHP-1 phosphatase activity counteracts increased T cell receptor affinity.

Anti-self/tumor T cell function can be improved by increasing TCR-peptide MHC (pMHC) affinity within physiological limits, but paradoxically further increases (K(d) < 1 μM) lead to drastic functional declines. Using human CD8(+) T cells engineered with TCRs of incremental affinity for the tumor antigen HLA-A2/NY-ESO-1, we investigated the molecular mechanisms underlying this high-affinity-associated loss of function. As compared with cells expressing TCR affinities generating optimal function (K(d) = 5 to 1 μM), those with supraphysiological affinity (K(d) = 1 μM to 15 nM) showed impaired gene expression, signaling, and surface expression of activatory/costimulatory receptors. Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade. In contrast, upregulation of the Src homology 2 domain-containing phosphatase 1 (SHP-1/PTPN6) was broad, with gradually enhanced expression in CD8(+) T cells with increasing TCR affinities. Consequently, pharmacological inhibition of SHP-1 with sodium stibogluconate augmented the function of all engineered T cells, and this correlated with the TCR affinity-dependent levels of SHP-1. These data highlight an unexpected and global role of SHP-1 in regulating CD8(+) T cell activation and responsiveness and support the development of therapies inhibiting protein tyrosine phosphatases to enhance T cell-mediated immunity.

Immune monitoring design within the developmental pipeline for an immunotherapeutic or preventive vaccine

Immunotherapy is defined as the treatment of disease by inducing, enhancing, or suppressing an immune response, whereas preventive vaccination is intended to prevent the development of diseases in healthy subjects. Most successful prophylactic vaccines rely on the induction of high titers of neutralizing antibodies. It is generally thought that therapeutic vaccination requires induction of robust T-cell mediated immunity. The diverse array of potential or already in use immunotherapeutic and preventive agents all share the commonality of stimulating the immune system. Hence, measuring those vaccination-induced immune responses gives the earliest indication of vaccine take and its immune modulating effects.

Female-biased mortality in experimentally parasitized Alpine Swift Apus melba nestlings

1. Sex-biased mortality in adult vertebrates is often attributed to lower immunocompetence and higher parasite susceptibility of males. Although sex-specific mortality has also been reported during growth, the importance of sex-specific immunocompetence and parasite susceptibility in explaining male-biased mortality remains ambiguous in growing individuals because of potentially confounding sources of mortality such as sexual dimorphism. 2. Here, we investigated sex-specific susceptibility to the blood-sucking louse fly Crataerina melbae and sex differences in cell-mediated immunity in a bird species that is sexually monomorphic both in size and plumage coloration at the nestling stage, the Alpine Swift, Apus melba. 3. For this purpose, we manipulated ectoparasite loads by adding or removing flies to randomly chosen nests in two years, and injected nestlings with mitogenic phytohaemagglutinin (PHA) in another year. 4. There were no significant differences between male and female offspring in immune response towards PHA, parasite load, and parasite-induced decrease in growth rate. Secondary sex ratios were however biased toward males in parasitized broods, and this was explained by a greater mortality of females in parasitized than deparasitized broods. 5. Our findings are in contrast to the widely accepted hypothesis that males suffer a greater cost of parasitism. We discuss alternative hypotheses accounting for female-specific mortality.

The synthetic Plasmodium falciparum circumsporozoite peptide PfCS102 as a malaria vaccine candidate: a randomized controlled phase I trial.

BACKGROUND: Fully efficient vaccines against malaria pre-erythrocytic stage are still lacking. The objective of this dose/adjuvant-finding study was to evaluate the safety, reactogenicity and immunogenicity of a vaccine candidate based on a peptide spanning the C-terminal region of Plasmodium falciparum circumsporozoite protein (PfCS102) in malaria naive adults. METHODOLOGY AND PRINCIPAL FINDINGS: Thirty-six healthy malaria-naive adults were randomly distributed into three dose blocks (10, 30 and 100 microg) and vaccinated with PfCS102 in combination with either Montanide ISA 720 or GSK proprietary Adjuvant System AS02A at days 0, 60, and 180. Primary end-point (safety and reactogenicity) was based on the frequency of adverse events (AE) and of abnormal biological safety tests; secondary-end point (immunogenicity) on P. falciparum specific cell-mediated immunity and antibody response before and after immunization. The two adjuvant formulations were well tolerated and their safety profile was good. Most AEs were local and, when systemic, involved mainly fatigue and headache. Half the volunteers in AS02A groups experienced severe AEs (mainly erythema). After the third injection, 34 of 35 volunteers developed anti-PfCS102 and anti-sporozoite antibodies, and 28 of 35 demonstrated T-cell proliferative responses and IFN-gamma production. Five of 22 HLA-A2 and HLA-A3 volunteers displayed PfCS102 specific IFN-gamma secreting CD8(+) T cell responses. Responses were only marginally boosted after the 3(rd) vaccination and remained stable for 6 months. For both adjuvants, the dose of 10 microg was less immunogenic in comparison to 30 and 100 microg that induced similar responses. AS02A formulations with 30 microg or 100 microg PfCS102 induced about 10-folds higher antibody and IFN-gamma responses than Montanide formulations. CONCLUSIONS/SIGNIFICANCE: PfCS102 peptide was safe and highly immunogenic, allowing the design of more advanced trials to test its potential for protection. Two or three immunizations with a dose of 30 microg formulated with AS02A appeared the most appropriate choice for such studies. TRIAL REGISTRATION: Swissmedic.ch 2002 DR 1227.

Clinical Experience with Immune Monitoring for Cytomegalovirus in Solid-Organ Transplant Recipients.

Novel strategies are needed to further reduce the burden of cytomegalovirus (CMV) disease in solid-organ transplant (SOT) recipients. Measurement of the specific cell-mediated immunity against CMV can identify the actual risk for the development of CMV disease in a given patient. Thus, immune monitoring is an attractive strategy for individualizing the management of CMV after transplantation. A growing number of observational studies on immune monitoring for CMV have been published over recent years, although there is a lack of data coming from interventional trials. In high-risk patients, measurement of CMV-specific T-cell responses appropriately stratifies the risk of CMV disease after discontinuation of antiviral prophylaxis. Immune monitoring may also help to identify patients followed by the preemptive approach at low risk for progression to CMV disease. Pretransplant assessment of cell-mediated immunity in seropositive patients may predict the development of posttransplant CMV infection. Overall, these studies indicate that the use of cell-mediated immunity assays has the potential to improve the management of CMV disease in SOT recipients.

Simultaneous coexpression of memory-related and effector-related genes by individual human CD8 T cells depends on antigen specificity and differentiation.

Phenotypic and functional cell properties are usually analyzed at the level of defined cell populations but not single cells. Yet, large differences between individual cells may have important functional consequences. It is likely that T-cell-mediated immunity depends on the polyfunctionality of individual T cells, rather than the sum of functions of responding T-cell subpopulations. We performed highly sensitive single-cell gene expression profiling, allowing the direct ex vivo characterization of individual virus-specific and tumor-specific T cells from healthy donors and melanoma patients. We have previously shown that vaccination with the natural tumor peptide Melan-A-induced T cells with superior effector functions as compared with vaccination with the analog peptide optimized for enhanced HLA-A*0201 binding. Here we found that natural peptide vaccination induced tumor-reactive CD8 T cells with frequent coexpression of both memory/homing-associated genes (CD27, IL7R, EOMES, CXCR3, and CCR5) and effector-related genes (IFNG, KLRD1, PRF1, and GZMB), comparable with protective Epstein-Barr virus-specific and cytomegalovirus-specific T cells. In contrast, memory/homing-associated and effector-associated genes were less frequently coexpressed after vaccination with the analog peptide. Remarkably, these findings reveal a previously unknown level of gene expression diversity among vaccine-specific and virus-specific T cells with the simultaneous coexpression of multiple memory/homing-related and effector-related genes by the same cell. Such broad functional gene expression signatures within antigen-specific T cells may be critical for mounting efficient responses to pathogens or tumors. In summary, direct ex vivo high-resolution molecular characterization of individual T cells provides key insights into the processes shaping the functional properties of tumor-specific and virus-specific T cells.

Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4(+) T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4(+) T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+) T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+) T cells and forkhead box P3 (FoxP3)(+) regulatory T (Treg) cells. IFN-γ produced mainly by CD4(+) T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.