[Mathematical Modeling of the Intracellular Regulation of Immune Processes].

The modern era of research in immunology is characterized by an unprecedented level of detail about structural characteristics of the immune system and the regulation of activities of its numerous components, which function together as a whole distributed-parameter system. Mathematical modeling provides an analytical tool to describe, analyze, and predict the dynamics of immune responses by applying a reductionist approach. In modern systems immunology and mathematical immunology as a new interdisciplinary field, a great challenge is to formulate the mathematical models of the human immune system that reflect the level achieved in understanding its structure and describe the processes that sustain its function. To this end, a systematic development of multiscale mathematical models has to be advanced. An appropriate methodology should consider (1) the intracellular processes of immune cell fate regulation, (2) the population dynamics of immune cells in various organs, and (3) systemic immunophysiological processes in the whole host organism. Main studies aimed at modeling the intracellular regulatory networks are reviewed in the context of multiscale mathematical modelling. The processes considered determine the regulation of the immune cell fate, including activation, division, differentiation, apoptosis, and migration. Because of the complexity and high dimensionality of the regulatory networks, identifying the parsimonious descriptions of signaling pathways and regulatory loops is a pressing problem of modern mathematical immunology.

Cancer immunotherapy of patients with HIV infection.

Cancer immunotherapy with antibodies against immune checkpoints has made impressive advances in the last several years. The most relevant drugs target programmed cell death 1 (PD-1) expressed on T cells or its ligand, the programmed cell death ligand 1 (PD-L1), expressed on cancer cells, and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Unfortunately, cancer patients with HIV infection are usually excluded from cancer clinical trials, because there are concerns about the safety and the anti-tumoral activity of these novel therapies in patients with HIV infection. Several retrospective studies and some case reports now support the notion that antibodies against immune checkpoints are safe and active in cancer patients with HIV infection, but prospective data in these patients are lacking. In addition, signs of antiviral activity with increase in CD4 T cell counts, plasma viremia reduction or decrease in the viral reservoir have been reported in some of the patients treated, although no patient achieved a complete clearance of the viral reservoir. Here we briefly summarize all clinical cases reported in the literature, as well as ongoing clinical trials testing novel immunotherapy drugs in cancer patients with HIV infection.

Interplay between reaction and diffusion processes in governing the dynamics of virus infections.

Spreading of viral infection in the tissues such as lymph nodes or spleen depends on virus multiplication in the host cells, their transport and on the immune response. Reaction-diffusion systems of equations with delays in cell proliferation and death by apoptosis represent an appropriate model to study this process. The properties of the cells of the immune system and the initial viral load determine the spatiotemporal regimes of infection spreading. Infection can be completely eliminated or it can persist at some level together with a certain chronic immune response in a spatially uniform or oscillatory mode. Finally, the immune cells can be completely exhausted leading to a high viral load persistence in the tissue. It has been found experimentally, that virus proteins can affect the immune cell migration. Our study shows that both the motility of immune cells and the virus infection spreading represented by the diffusion rate coefficients are relevant control parameters determining the fate of virus-host interaction.

Towards standardized automated immunomonitoring: an automated ELISpot assay for safe and parallelized functionality analysis of immune cells.

The ELISpot assay is used for the detection of T cell responses in clinical trials and vaccine evaluations. Standardization and reproducibility are necessary to compare the results worldwide, inter- and intra-assay variability being critical factors. To assure operator safety as well as high-quality experiment performance, the ELISpot assay was implemented on an automated liquid handling platform, a Tecan Freedom EVO. After validation of the liquid handling, automated loading of plates with cells and reagents was investigated. With step by step implementation of the manual procedure and liquid dispensing optimization on the robot platform, a fully automated ELISpot assay was accomplished with plates remaining in the system from the plate blocking step to spot development. The mean delta difference amounted to a maximum of 6%, and the mean dispersion was smaller than in the manual assay. Taken together, we achieved with this system not only a lower personnel attendance but also higher throughput and a more precise and parallelized analysis. This platform has the potential to guarantee validated, safe, fast, reproducible and cost-efficient immunological and toxicological assays in the future.

Detection of Broadly Neutralizing Activity within the First Months of HIV-1 Infection.

UNLABELLED: A fraction of HIV-1 patients are able to generate broadly neutralizing antibodies (bNAbs) after 2 to 4 years of infection. In rare occasions such antibodies are observed close to the first year of HIV-1 infection but never within the first 6 months. In this study, we analyzed the neutralization breadth of sera from 157 antiretroviral-naive individuals who were infected for less than 1 year. A range of neutralizing activities was observed with a previously described panel of six recombinant viruses from five different subtypes (M. Medina-Ramirez et al., J Virol 85:5804-5813, 2011, http://dx.doi.org/10.1128/JVI.02482-10). Some sera were broadly reactive, predominantly targeting envelope epitopes within the V2 glycan-dependent region. The neutralization breadth was positively associated with time postinfection (P = 0.0001), but contrary to what has been reported for chronic infections, no association with the viral load was observed. Notably, five individuals within the first 6 months of infection (two as early as 77 and 96 days postinfection) showed substantial cross-neutralization. This was confirmed with an extended panel of 20 Env pseudoviruses from four different subtypes (two in tier 3, 14 in tier 2, and four in tier 1). Sera from these individuals were capable of neutralizing viruses from four different subtypes with a geometric mean 50% infective dose (ID50) between 100 and 800. These results indicate that induction of cross-neutralizing responses, albeit rare, is achievable even within 6 months of HIV-1 infection. These observations encourage the search for immunogens able to elicit this kind of response in preventive HIV-1 vaccine approaches. IMPORTANCE: There are very few individuals able to mount broadly neutralizing activity (bNA) close to the first year postinfection. It is not known how early in the infection cross-neutralizing responses can be induced. In the present study, we show that bNAbs, despite being rare, can be induced much earlier than previously thought. The identification of HIV-1-infected patients with these activities within the first months of infection and characterization of these responses will help in defining new immunogen designs and neutralization targets for vaccine-mediated induction of bNAbs.

Antiviral drug discovery: broad-spectrum drugs from nature.

Covering: up to April 2014. The development of drugs with broad-spectrum antiviral activities is a long pursued goal in drug discovery. It has been shown that blocking co-opted host-factors abrogates the replication of many viruses, yet the development of such host-targeting drugs has been met with scepticism mainly due to toxicity issues and poor translation to in vivo models. With the advent of new and more powerful screening assays and prediction tools, the idea of a drug that can efficiently treat a wide range of viral infections by blocking specific host functions has re-bloomed. Here we critically review the state-of-the-art in broad-spectrum antiviral drug discovery. We discuss putative targets and treatment strategies, with particular focus on natural products as promising starting points for antiviral lead development.

Pathogenesis and treatment of HIV infection: the cellular, the immune system and the neuroendocrine systems perspective.

Infections with HIV represent a great challenge for the development of strategies for an effective cure. The spectrum of diseases associated with HIV ranges from opportunistic infections and cancers to systemic physiological disorders like encephalopathy and neurocognitive impairment. A major progress in controlling HIV infection has been achieved by highly active antiretroviral therapy (HAART). However, HAART does neither eliminate the virus reservoirs in form of latently infected cells nor does it completely reconstitute immune reactivity and physiological status. Furthermore, the failure of the STEP vaccine trial and the only marginal efficacies of the RV144 trial together suggest that the causal relationships between the complex sets of viral and immunological processes that contribute to protection or disease pathogenesis are still poorly understood. Here, we provide an up-to-date overview of HIV-host interactions at the cellular, the immune system and the neuroendocrine systems level. Only by integrating this multi-level knowledge one will be able to handle the systems complexity and develop new methodologies of analysis and prediction for a functional restoration of the immune system and the health of the infected host.

Challenges and perspectives for improved management of HIV/Mycobacterium tuberculosis co-infection.

HIV and Mycobacterium tuberculosis (MTB) are two widespread and highly successful microbes whose synergy in pathogenesis has created a significant threat for human health globally. In acknowledgement of this fact, the European Union (EU) has funded a multinational support action, the European Network for global cooperation in the field of AIDS and TB (EUCO-Net), that brings together experts from Europe and those regions that bear the highest burden of HIV/MTB co-infection. Here, we summarise the main outcome of the EUCO-Net project derived from an expert group meeting that took place in Stellenbosch (South Africa) (AIDS/TB Workshop on Research Challenges and Opportunities for Future Collaboration) and the subsequent discussions, and propose priority areas for research and concerted actions that will have impact on future EU calls.

PROCAM-, FRAMINGHAM-, SCORE- and SMART-risk score for predicting cardiovascular morbidity and mortality in patients with overt atherosclerosis.

BACKGROUND: The predictive value of PROCAM, FRAMINGHAM, SCORE and SMART-score to estimate the cardiovascular risk in patients with overt atherosclerosis had never been assessed. PATIENTS AND METHODS: 96 consecutive patients with clinically evident atherosclerosis (coronary, cerebrovascular, peripheral artery and renovascular disease) were enrolled in this preliminary observational study. At baseline, medical history and blood chemistry were obtained. Sonographic measurement of the intima-media thickness (IMT) in the common carotid artery was performed and risk estimations according to the above listed risk scores were calculated. During a 6 year follow-up the occurrence of cardiovascular death, acute coronary syndrome and stroke was assessed. RESULTS: Mean (±SD) risk-scores were 10.9±2.5, range 6-17 (SMART); 18.9±18.2%; range 0.2-94.1% (PROCAM); 21.4±13.1%, range 4-56% (FRAMINGHAM); and 4.8±3.9%, range 0.4-15.3% (SCORE). Mean IMT was 0.84±0.14 mm, range 0.51-1.20 mm. All scores correlate significantly with each other (r>0.321; p<0.01), but only SMART-score correlated significantly with baseline IMT(r=0.372; p<0.001). Within the median follow-up of 73 months, a cardiovascular endpoint was observed in 36 (42%) patients. The AUC (95% confidence interval) for SMART-risk-score predicting a cardiovascular event was 0.67 (0.54-0.77; p<0.02); for PROCAM 0.60 (0.47-0.73; p=n.s.); for FRAMINGHAM 0.56 (0.43-0.69; p=n.s.); and for SCORE 0.60 (0.46-0.73; p=n.s.). Cox regression analysis showed a relative risk for a cardiovascular event per additional SMART score point of 1.15 (95% CI 1.01-1.30 p=0.03). CONCLUSIONS: PROCAM-, FRAMINGHAM- and SCORE-risk score seem to be barely useful in a secondary prevention setting. In patients with overt atherosclerosis, the cardiovascular risk seems to be better assessed by means of the SMART score.

Intima-media thickness and carotid resistive index: progression over 6 years and predictive value for cardiovascular events.

PURPOSE: Intima-media thickness (IMT) of the common carotid artery and the resistive index (RI) of the internal carotid artery correlate with the degree of atherosclerosis and are predictors of cardiovascular morbidity and mortality. Limited or no data are available about long-term predictive values and the progression of the two markers themselves. MATERIALS AND METHODS: 145 patients with at least one cardiovascular risk factor or clinically manifest atherosclerosis were included. At enrollment and after 36 and 74 months, duplex sonographic measurements of IMT CCA and RI ICA were performed. During follow-up, the occurrence of cardiovascular events (cardiovascular death, myocardial infarction, stroke) was assessed. RESULTS: At baseline, IMT was 0.79 +/- 0.16 mm and RI 0.66 +/- 0.08. Log-rank analysis showed a continuous increase in the risk of a cardiovascular event with an increasing range of IMT (p = 0.011) and RI (p = 0.006). IMT progression in patients with low versus high atherosclerotic burden (as defined by SMART score < or =7 points and > 7 points) differs significantly (32 +/- 83 microm versus 95 +/- 125 microm; p < 0.002). IMT progression was even more pronounced in patients suffering a cardiovascular event (141 +/- 105 microm versus 54 +/- 111 microm; p < 0.001). No significant RI ICA progression could be detected during follow-up in any group (patients with low vs. high atherosclerotic burden 0.00 +/- 0.06 versus 0.00 +/- 0.04; p = n. s.; patients with vs. without cardiovascular event 0.00 +/- 0.05 versus 0.01 +/- 0.03; p = n. s.). CONCLUSION: Our results confirm the predictive value for cardiovascular events of RI and IMT in long-term follow-up. In contrast to RI, IMT increases over six years, above all in patients suffering a cardiovascular event. The results suggest that IMT is suitable for cardiovascular risk prediction as well as for progression measurements, while RI cannot be recommended for progression measurements. The effect of drug therapy on RI needs further clarification.

Human cytomegalovirus protein pp65: an efficient protein carrier system into human dendritic cells.

Protein-based immunogens are usually poor inducers of CD8(+) T cells. To enhance the induction of CD8(+) T cells, one approach is the use of protein immunogens coupled to protein transduction domains (PTDs). These are small cationic peptide sequences that significantly enhance the uptake of fused proteins into dendritic cells (DC) and then mediate their presentation in the context of major histocompatibility complex class I (MHC-I) and MHC-II molecules. One drawback of this system is the high concentrations of PTD-fusion proteins required. Here, we show that proteins fused to the human cytomegalovirus tegument protein pp65 were bound with higher efficiency to DCs than those fused to the described PTDs TatPTD and Penetratin. Furthermore, the fusion of pp65 to proteins led to an enhanced uptake of these proteins by DCs. Once taken up, CD4(+) and CD8(+) memory T cells were strongly stimulated ex vivo demonstrating that pp65 was efficiently processed and presented in the context of both MHC-I and MHC-II. These data make pp65 a promising delivery system to induce cellular immune responses by fused protein vaccines.

Levels of virus-specific CD4 T cells correlate with cytomegalovirus control and predict virus-induced disease after renal transplantation.

BACKGROUND: Immunosuppressive treatment in transplant patients frequently causes infectious complications with cytomegalovirus (CMV). The extent of CMV replication can be followed by a number of diagnostic methods. There is, however, no simple diagnostic tool to assess the quality of the cellular antiviral immune response of an individual patient. This would be of particular importance for therapy decisions, as patients with detectable virus load do not necessarily develop CMV-related disease. Using a rapid whole blood assay, the frequencies of CMV-reactive CD4 and CD8 T cells were followed after renal transplantation to characterize their relative contribution in the containment of CMV infection. METHODS: T cells from transplant patients ands healthy control persons were stimulated with CMV antigen in vitro. Based on specific cellular activation and induction of intracellular cytokines, the frequency of CMV-reactive CD4 and CD8 T cells was determined using flow cytometry. Viral load quantified using the "hybrid-capture" assay. RESULTS: The absence of CMV complications in long-term transplant recipients is reflected by stable virus-specific T-cell frequencies, which do not differ from healthy CMV-positive controls. In contrast, during the first months after transplantation, clinical symptoms are preceded by a decrease in CMV-reactive CD4 T-cell frequencies and an increase in CMV load. CONCLUSIONS: The individual immune response and CMV replication are critically balanced and can be characterized by assesing both viral load and antiviral T cells. Our experimental design allows the identification of patients with sufficient, insufficient, or absent T-cell activity and can serve as diagnostic tool to facilitate decisions on antiviral therapy.

Rapid increase of mucosal CD4 T cells followed by clearance of intestinal cryptosporidiosis in an AIDS patient receiving highly active antiretroviral therapy.

Highly active antiretroviral therapy (HAART) suppresses the replication of human immunodeficiency virus (HIV) and leads to an increase in circulating CD4 T lymphocytes, but its effects on other immune compartments such as the intestinal mucosa are not well understood. We describe a severely immunodeficient HIV-infected patient with intractable watery diarrhea and weight loss caused by infection with Cryptosporidium parvum in whom we studied virologic and immunologic changes in both peripheral blood and the intestinal mucosa after initiating HAART. Mucosal biopsies were performed by rectoscopy before and at several time points after HAART was begun. Nucleic acids were extracted from rectal biopsy specimens and blood samples, and HIV RNA was measured by reverse-transcription polymerase chain reaction. Lymphocytes were isolated from rectal biopsy specimens after mechanical disaggregation, and circulating and mucosal CD4 T cells were determined by flow cytometry. HAART led to clinical recovery and eradication of cryptosporidiosis. In both blood and mucosa, HIV RNA decreased below the limit of detection and CD4 T cells increased. Mucosal CD4 T cells increased much faster and to much higher levels than circulating CD4 T cells. Our findings show a rapid repopulation of the intestinal mucosa with CD4 T cells after initiation of HAART that can effectively restore mucosal immunity, leading to eradication of opportunistic pathogens.

Rapid whole blood analysis of virus-specific CD4 and CD8 T cell responses in persistent HIV infection.

OBJECTIVES: Upon HIV infection, strong antiviral cytotoxic and helper T cell responses are generated. They are considered to be an important component in the control of HIV viral load. A simple and rapid whole blood assay was established to quantify and simultaneously characterize HIV-reactive CD4 and CD8 cells. The assay was applied to evaluate the effect of antiretroviral therapy on HIV-specific T cell responses. METHODS: Whole blood of 33 HIV-infected individuals was specifically stimulated by HIV-1 Pr55gag, and activation-induced intracellular cytokine expression in CD4 and CD8 T cells was analysed by flow cytometry. RESULTS: HIV-1-specific CD8 and CD4 T cells can be quantified simultaneously. As specific antigen, HIV-1 Pr55gag virus-like particles were superior to soluble protein, especially for the activation of CD8 T cells. In untreated individuals, a high frequency of HIV-specific T cells was observed. The frequency of CD8 T cells was consistently higher than the respective CD4 T cell response, thus demonstrating a dominance in CD8 T cell expansion in persistent HIV infection. Patients on antiretroviral therapy showed a significant reduction in HIV-specific CD4 and, even more strikingly, CD8 T cells. CONCLUSION: The whole blood assay provides a rapid estimate of the total antiviral T cell resources, and is highly suited for a clinical setting. It may thus have widespread applications for the evaluation of vaccination strategies and immunotherapy. Because antiretroviral therapy significantly reduces both HIV-specific cytotoxic and helper T cell responses, future therapeutic strategies should aim at improving cellular antiviral immunity.

Kinetics of CXCR4 and CCR5 up-regulation and human immunodeficiency virus expansion after antigenic stimulation of primary CD4(+) T lymphocytes.

The chemokine receptors CCR5 and CXCR4 are coreceptors for the human immunodeficiency virus (HIV) and determine the cell tropism of different HIV strains. Previous studies on their regulation were performed under conditions of unspecific T-lymphocyte stimulation and provided conflicting results. To mimic physiologic conditions, highly purified primary Staphylococcus enterotoxin B (SEB)-reactive CD4 T lymphocytes were stimulated in the presence of autologous antigen-presenting cells and the kinetics of CCR5 and CXCR4 surface expression and HIV replication were studied. Both chemokine receptors were transiently up-regulated with maximal expression at day 3 after stimulation. The stimulated T cells were equally susceptible to productive infection with R5-and X4-tropic virus strains. Thus, antigenic stimulation of T cells promotes efficient replication of both, T cell-tropic and macrophage-tropic HIV. (Blood. 2000;96:1853-1856)

Cytotoxic T lymphocyte epitopes of HIV-1 Nef: Generation of multiple definitive major histocompatibility complex class I ligands by proteasomes.

Although a pivotal role of proteasomes in the proteolytic generation of epitopes for major histocompatibility complex (MHC) class I presentation is undisputed, their precise function is currently the subject of an active debate: do proteasomes generate many epitopes in definitive form, or do they merely generate the COOH termini, whereas the definitive NH(2) termini are cleaved by aminopeptidases? We determined five naturally processed MHC class I ligands derived from HIV-1 Nef. Unexpectedly, the five ligands correspond to only three cytotoxic T lymphocyte (CTL) epitopes, two of which occur in two COOH-terminal length variants. Parallel analyses of proteasomal digests of a Nef fragment encompassing the epitopes revealed that all five ligands are direct products of proteasomes. Moreover, in four of the five ligands, the NH(2) termini correspond to major proteasome cleavage sites, and putative NH(2)-terminally extended precursor fragments were detected for only one of the five ligands. All ligands are transported by the transporter associated with antigen processing (TAP). The combined results from these five ligands provide strong evidence that many definitive MHC class I ligands are precisely cleaved at both ends by proteasomes. Additional evidence supporting this conclusion is discussed, along with contrasting results of others who propose a strong role for NH(2)-terminal trimming with direct proteasomal epitope generation being a rare event.

Gamma interferon is a major suppressive factor produced by activated human peripheral blood lymphocytes that is able to inhibit foamy virus-induced cytopathic effects.

The activation of human peripheral blood lymphocytes by mitogens or by triggering the T-cell receptor with anti-CD3 antibodies leads to the production of a potent soluble inhibitory activity against foamy virus-induced cytopathic effects in vitro. The inhibitory activity acts in a species-specific manner. As a consequence, the isolation of foamy viruses from blood lymphocytes of infected humans is accelerated in a heterologous coculture system. Antibodies against gamma interferon (IFN-gamma) are able to suppress most of the inhibitory activity, suggesting that IFN-gamma is the dominant component.

On the role of the second coding exon of the HIV-1 Tat protein in virus replication and MHC class I downregulation.

Tat is an essential protein of human immunodeficiency virus type 1 (HIV-1) and activates transcription from the viral long terminal repeat (LTR) promoter. The tat gene is composed of two coding exons of which the first, corresponding to the N-terminal 72 amino acid residues, has been reported to be sufficient for its transcription function. We introduced a stop codon at the end of the first Tat-coding exon in an expression vector that produces a truncated 71-amino acid Tat protein. This Q72stop mutant displays reduced transcriptional activity of approximately 54% in transient LTR-CAT transfection assays. To test the contribution of the second Tat-coding exon to virus replication, the Q72stop mutation was also introduced in the infectious pLAI molecular clone. The effect on virus replication was analyzed in primary cells and in a transformed T cell line. The fitness of the mutant virus was calculated to be approximately 75% compared with the wild-type control. Thus, a small contribution of the C-terminal Tat domain to viral fitness was measured. It has been proposed that the second Tat-coding exon is involved in transcriptional downregulation of the MHC class I gene of the infected host cell. Cell surface expression of the MHC protein was analyzed in T cells infected with the wild-type LAI virus and the replication-competent Q72stop mutant. MHC expression was transiently reduced on infection with either virus, indicating that the second Tat-coding exon is not involved in this downregulation.