Multiple transmission routes sustain high prevalence of a virulent parasite in a butterfly host.

Understanding factors that allow highly virulent parasites to reach high infection prevalence in host populations is important for managing infection risks to human and wildlife health. Multiple transmission routes have been proposed as one mechanism by which virulent pathogens can achieve high prevalence, underscoring the need to investigate this hypothesis through an integrated modelling-empirical framework. Here, we examine a harmful specialist protozoan infecting monarch butterflies that commonly reaches high prevalence (50-100%) in resident populations. We integrate field and modelling work to show that a combination of three empirically-supported transmission routes (vertical, adult transfer and environmental transmission) can produce and sustain high infection prevalence in this system. Although horizontal transmission is necessary for parasite invasion, most new infections post-establishment arise from vertical transmission. Our study predicts that multiple transmission routes, coupled with high parasite virulence, can reduce resident host abundance by up to 50%, suggesting that the protozoan could contribute to declines of North American monarchs.

Increasing inflationary T-cell responses following transient depletion of MCMV-specific memory T cells.

Murine CMV (MCMV) infection induces effector CD8(+) T cells that continue to increase in frequency after acute infection ("inflation") and are stably maintained at a high frequency, with up to 20% of the CD8(+) T-cell compartment being specific for one epitope, although the flexibility and turnover of these populations is not fully defined. Here we report that effector/memory CD8(+) T cells induced by MCMV can be paradoxically boosted following transient depletion of epitope specific CD8(+) T cells. Treatment of MCMV-infected mice with MHC-Class I-saporin tetramers led to partial (80-90%) depletion of epitope-specific CD8(+) T cells-rapidly followed by a rebound, leading to expansion and maintenance of up to 40% of total CD8(+) T cells, with minimal changes in response to a control epitope (M45). These data indicate the tight balance between host and virus during persistent infection and the functional flexibility of the "inflated" CD8(+) T cell responses during persistent infection.

A new model for CD8+ T cell memory inflation based upon a recombinant adenoviral vector.

CD8(+) T cell memory inflation, first described in murine CMV (MCMV) infection, is characterized by the accumulation of high-frequency, functional Ag-specific CD8(+) T cell pools with an effector-memory phenotype and enrichment in peripheral organs. Although persistence of Ag is considered essential, the rules underpinning memory inflation are still unclear. The MCMV model is, however, complicated by the virus's low-level persistence and stochastic reactivation. We developed a new model of memory inflation based on a ß-galactosidase (ßgal)-recombinant adenovirus vector. After i.v. administration in C57BL/6 mice, we observed marked memory inflation in the ßgal96 epitope, whereas a second epitope, ßgal497, undergoes classical memory formation. The inflationary T cell responses show kinetics, distribution, phenotype, and functions similar to those seen in MCMV and are reproduced using alternative routes of administration. Memory inflation in this model is dependent on MHC class II. As in MCMV, only the inflating epitope showed immunoproteasome independence. These data define a new model for memory inflation, which is fully replication independent, internally controlled, and reproduces the key immunologic features of the CD8(+) T cell response. This model provides insight into the mechanisms responsible for memory inflation and, because it is based on a vaccine vector, also is relevant to novel T cell-inducing vaccines in humans.

Age-associated increase of low-avidity cytomegalovirus-specific CD8+ T cells that re-express CD45RA.

The mechanisms regulating memory CD8(+) T cell function and homeostasis during aging are unclear. CD8(+) effector memory T cells that re-express CD45RA increase considerably in older humans and both aging and persistent CMV infection are independent factors in this process. We used MHC class I tetrameric complexes that were mutated in the CD8 binding domain to identify CMV-specific CD8(+) T cells with high Ag-binding avidity. In individuals who were HLA-A*0201, CD8(+) T cells that expressed CD45RA and were specific for the pp65 protein (NLVPMVATV epitope) had lower avidity than those that expressed CD45RO and demonstrated decreased cytokine secretion and cytolytic potential after specific activation. Furthermore, low avidity NLVPMVATV-specific CD8(+) T cells were significantly increased in older individuals. The stimulation of blood leukocytes with CMV lysate induced high levels of IFN-α that in turn induced IL-15 production. Moreover, the addition of IL-15 to CD45RA(-)CD45RO(+) CMV-specific CD8(+) T cells induced CD45RA expression while Ag activated cells remained CD45RO(+). This raises the possibility that non-specific cytokine-driven accumulation of CMV-specific CD8(+)CD45RA(+) T cells with lower Ag-binding avidity may exacerbate the effects of viral reactivation on skewing the T cell repertoire in CMV-infected individuals during aging.

A genetic and functional relationship between T cells and cellular proliferation in the adult hippocampus.

Neurogenesis continues through the adult life of mice in the subgranular zone of the dentate gyrus in the hippocampus, but its function remains unclear. Measuring cellular proliferation in the hippocampus of 719 outbred heterogeneous stock mice revealed a highly significant correlation with the proportions of CD8+ versus CD4+ T lymphocyte subsets. This correlation reflected shared genetic loci, with the exception of the H-2Ea locus that had a dominant influence on T cell subsets but no impact on neurogenesis. Analysis of knockouts and repopulation of TCRα-deficient mice by subsets of T cells confirmed the influence of T cells on adult neurogenesis, indicating that CD4+ T cells or subpopulations thereof mediate the effect. Our results reveal an organismal impact, broader than hitherto suspected, of the natural genetic variation that controls T cell development and homeostasis.

High frequency, sustained T cell responses to PARV4 suggest viral persistence in vivo.

BACKGROUND: Parvovirus 4 (PARV4) is a recently identified human virus that has been found in livers of patients infected with hepatitis C virus (HCV) and in bone marrow of individuals infected with human immunodeficiency virus (HIV). T cells are important in controlling viruses but may also contribute to disease pathogenesis. The interaction of PARV4 with the cellular immune system has not been described. Consequently, we investigated whether T cell responses to PARV4 could be detected in individuals exposed to blood-borne viruses. METHODS: Interferon γ (IFN-γ) enzyme-linked immunospot assay, intracellular cytokine staining, and a tetrameric HLA-A*0201-peptide complex were used to define the lymphocyte populations responding to PARV4 NS peptides in 88 HCV-positive and 13 HIV-positive individuals. Antibody responses were tested using a recently developed PARV4 enzyme-linked immunosorbent assay. RESULTS: High-frequency T cell responses against multiple PARV4 NS peptides and antibodies were observed in 26% of individuals. Typical responses to the NS pools were >1000 spot-forming units per million peripheral blood mononuclear cells. CONCLUSIONS: PARV4 infection is common in individuals exposed to blood-borne viruses and elicits strong T cell responses, a feature typically associated with persistent, contained infections such as cytomegalovirus. Persistence of PARV4 viral antigen in tissue in HCV-positive and HIV-positive individuals and/or the associated activated antiviral T cell response may contribute to disease pathogenesis.

Molecular footprints reveal the impact of the protective HLA-A*03 allele in hepatitis C virus infection.

BACKGROUND AND AIMS: CD8 T cells are central to the control of hepatitis C virus (HCV) although the key features of a successful CD8 T cell response remain to be defined. In a cohort of Irish women infected by a single source, a strong association between viral clearance and the human lecucocyte (HLA)-A*03 allele has been described, and the aim of this study was to define the protective nature of the associated CD8 T cell response. METHODS: A sequence-led approach was used to identify HLA-A*03-restricted epitopes. We examine the CD8 T cell response associated with this gene and address the likely mechanism underpinning this protective effect in this special cohort, using viral sequencing, T cell assays and analysis of fitness of viral mutants. RESULTS: A strong 'HLA footprint' in a novel NS3 epitope (TVYHGAGTK) was observed. A lysine (K) to arginine (R) substitution at position 9 (K1088R) was seen in a significant number of A*03-positive patients (9/12) compared with the control group (1/33, p=0.0003). Threonine (T) was also substituted with alanine (A) at position 8 (T1087A) more frequently in A*03-positive patients (6/12) compared with controls (2/33, p=0.01), and the double substitution of TK to AR was also observed predominantly in HLA-A*03-positive patients (p=0.004). Epitope-specific CD8 T cell responses were observed in 60% of patients three decades after exposure and the mutants selected in vivo impacted on recognition in vitro. Using HCV replicons matched to the viral sequences, viral fitness was found to be markedly reduced by the K1088R substitution but restored by the second substitution T1087A. CONCLUSIONS: It is proposed that at least part of the protective effect of HLA-A*03 results from targeting of this key epitope in a functional site: the requirement for two mutations to balance fitness and escape provides an initial host advantage. This study highlights the potential protective impact of common HLA-A alleles against persistent viruses, with important implications for HCV vaccine studies.

Real-life research projects improve student engagement and provide reliable data for academics.

Student engagement can have a positive influence on student success. Many methods exist for fostering engagement but tend to be generic and require tailoring to specific contexts, subjects, and students. In the case of undergraduate science students, practical classes are a popular tool for increasing engagement. However, despite strong potential for improvement via links with "real life" research projects (RLRPs), few academic staff incorporate research participation with teaching activities. This is potentially due to poor time availability and low opinions of students' ability to collect reliable data. This study aims to examine whether involvement with RLRPs can generate reliable scientific data and also act as a motivational tool for engaging tertiary science students. A preexisting core activity for first-year biology and marine biology students was modified to include a short RLRP component. Student-based data collection and a questionnaire about experiences were used to examine the reliability of student-collected data and student perceptions of RLRPs. Results indicated that error rate in student-collected data was minimal. Irrespective of participating in a "normal" practical class or a class with a RLRP component, students collected equally accurate data. However, when the topic aligned specifically with their degree subject, student accuracy was higher. All students surveyed reported high motivation with the idea of RLRP participation, placing high importance on this from an educational and employability perspective. Yet, students were not confident about participating in RLRPs until they had engaged with one, suggesting that introducing such projects into taught sessions early-on may encourage students to seek further opportunities in the future. In conclusion, incorporating RLRPs into the curriculum of undergraduate science courses has considerable potential benefits for both students and academic staff.

Defining the directionality and quality of influenza virus-specific CD8+ T cell cross-reactivity in individuals infected with hepatitis C virus.

Cross-reactivity of murine and recently human CD8(+) T cells between different viral peptides, i.e., heterologous immunity, has been well characterized. However, the directionality and quality of these cross-reactions is critical in determining their biological importance. Herein we analyzed the response of human CD8(+) T cells that recognize both a hepatitis C virus peptide (HCV-NS3) and a peptide derived from the influenza neuraminidase protein (Flu-NA). To detect the cross-reactive CD8(+) T cells, we used peptide-MHC class I complexes (pMHCs) containing a new mutant form of MHC class I able to bind CD8 more strongly than normal MHC class I complexes. T cell responses against HCV-NS3 and Flu-NA peptide were undetectable in normal donors. In contrast, some responses against the Flu-NA peptide were identified in HCV(+) donors who showed strong HCV-NS3-specific reactivity. The Flu-NA peptide was a weak agonist for CD8(+) T cells in HCV(+) individuals on the basis of novel pMHCs and functional assays. These data support the idea of cross-reactivity between the 2 peptides, but indicate that reactivity toward the Flu-NA peptide is highly CD8-dependent and occurs predominantly after priming during HCV infection. Our findings indicate the utility of the novel pMHCs in dissecting cross-reactivity and suggest that cross-reactivity between HCV and influenza is relatively weak. Further studies are needed to relate affinity and functionality of cross-reactive T cells.

HLArestrictor--a tool for patient-specific predictions of HLA restriction elements and optimal epitopes within peptides.

Traditionally, T cell epitope discovery requires considerable amounts of tedious, slow, and costly experimental work. During the last decade, prediction tools have emerged as essential tools allowing researchers to select a manageable list of epitope candidates to test from a larger peptide, protein, or even proteome. However, no current tools address the complexity caused by the highly polymorphic nature of the restricting HLA molecules, which effectively individualizes T cell responses. To fill this gap, we here present an easy-to-use prediction tool named HLArestrictor ( http://www.cbs.dtu.dk/services/HLArestrictor ), which is based on the highly versatile and accurate NetMHCpan predictor, which here has been optimized for the identification of both the MHC restriction element and the corresponding minimal epitope of a T cell response in a given individual. As input, it requires high-resolution (i.e., 4-digit) HLA typing of the individual. HLArestrictor then predicts all 8-11mer peptide binders within one or more larger peptides and provides an overview of the predicted HLA restrictions and minimal epitopes. The method was tested on a large dataset of HIV IFNγ ELIspot peptide responses and was shown to identify HLA restrictions and minimal epitopes for about 90% of the positive peptide/patient pairs while rejecting more than 95% of the negative peptide-HLA pairs. Furthermore, for 18 peptide/HLA tetramer validated responses, HLArestrictor in all cases predicted both the HLA restriction element and minimal epitope. Thus, HLArestrictor should be a valuable tool in any T cell epitope discovery process aimed at identifying new epitopes from infectious diseases and other disease models.

Efficacious early antiviral activity of HIV Gag- and Pol-specific HLA-B 2705-restricted CD8+ T cells.

The association between HLA-B 2705 and the immune control of human immunodeficiency virus type 1 (HIV-1) has previously been linked to the targeting of the HLA-B 2705-restricted Gag epitope KRWIILGLNK (KK10) by CD8(+) T cells. In order to better define the mechanisms of the HLA-B 2705 immune control of HIV, we first characterized the CD8(+) T-cell responses of nine highly active antiretroviral therapy (HAART)-naïve B 2705-positive subjects. Unexpectedly, we observed a strong response to an HLA-B 2705-restricted Pol epitope, KRKGGIGGY (KY9), in 8/9 subjects. The magnitude of the KY9 response was only marginally lower than that of the KK10-specific response (median, 695 versus 867 spot-forming cells [SFC]/million peripheral blood mononuclear cells [PBMCs]; not significant [NS]), and viral escape mutants were observed in both KY9 and KK10, resulting from selection pressure driven by the respective CD8(+) T-cell response. By comparing inhibitions of viral replication by CD8(+) T cells specific for the Gag KK10, Pol KY9, and Vpr VL9 HLA-B 2705-restricted epitopes, we observed a consistent hierarchy of antiviral efficacy (Gag KK10 > Pol KY9 > Vpr VL9). This hierarchy was associated with early recognition of HIV-1-infected cells, within 6 h of infection, by KK10- and KY9-specific CD8(+) T cells but not until 18 h postinfection by VL9-specific CD8(+) T cells. There was no association between antiviral efficacy and proliferative capacity, cytotoxicity, polyfunctionality, or T-cell receptor (TCR) avidity. These data are consistent with previous studies indicating an important role for the B 2705-Gag KK10 response in the control of HIV but also suggest a previously unrecognized role played by the subdominant Pol-specific KY9 response in HLA-B 2705-mediated control of HIV and that the recognition of HIV-infected cells by CD8(+) T cells early in the viral life cycle may be important for viral containment in HIV-infected individuals.

In Vitro Comparison of the Internal Ribosomal Entry Site Activity from Rodent Hepacivirus and Pegivirus and Construction of Pseudoparticles.

The 5' untranslated region (5' UTR) of rodent hepacivirus (RHV) and pegivirus (RPgV) contains sequence homology to the HCV type III internal ribosome entry sites (IRES). Utilizing a monocistronic expression vector with an RNA polymerase I promoter to drive transcription, we show cell-specific IRES translation and regions within the IRES required for full functionality. Focusing on RHV, we further pseudotyped lentivirus with RHV and showed cell surface expression of the envelope proteins and transduction of murine hepatocytes and we then constructed full-length RHV and RPgV replicons with reporter genes. Using the replicon system, we show that the RHV NS3-4A protease cleaves a mitochondrial antiviral signaling protein reporter. However, liver-derived cells did not readily support the complete viral life cycle.

Parasite Manipulation of Its Host's Physiological Reaction to Acute Stress: Experimental Results from a Natural Beetle-Nematode System.

All animals, whether vertebrate or invertebrate, must be capable of reacting to acute stressors, such as escaping from predators, and most do so with a suite of transient physiological changes that temporarily enhance survival. Some of these changes include mobilization of immune cells and increased cardiac output. A small but growing number of studies have begun to show that certain parasites appear capable of modifying such responses. We addressed this topic using a natural host and parasite system, that is, a nematode (Chondronema passali) that parasitizes horned passalus beetles, Odontotaenius disjunctus (family Passalidae), of the eastern United States. With a series of experiments, we sought to determine whether this parasite affects (1) the immune reaction to stress, (2) the output of stress-induced alarm calls, or (3) the increase in heart rate that occurs in response to acute stressors, with the stressors being mechanical or thermal. Results showed that hemocyte density increased after both stressors in nonparasitized beetles but did not increase in parasitized beetles. While mobilization of immune cells would enhance host immunity during stress, this would also be damaging to the nematode, so this scenario appears to benefit the parasite. We found no evidence that the nematode suppresses the overall reaction to stress (or prevents stress from occurring), since parasitized beetles did not differ from nonparasitized ones in alarm call rates or in heart beat frequency after exposure to mechanical stressors. Suppression of the host's normal immune reaction to stressful stimuli could translate to delayed or even reduced wound healing or pathogen resistance during these events. This project is a rare demonstration of parasite manipulation of host immune response to acute stress and should stimulate further investigations into the interactive nature of stress and parasites.

Saporin-conjugated tetramers identify efficacious anti-HIV CD8+ T-cell specificities.

Antigen-specific T-cells are highly variable, spanning potent antiviral efficacy and damaging auto-reactivity. In virus infections, identifying the most efficacious responses is critical to vaccine design. However, current methods depend on indirect measures or on ex vivo expanded CTL clones. We here describe a novel application of cytotoxic saporin-conjugated tetramers to kill antigen-specific T-cells without significant off-target effects. The relative efficacy of distinct antiviral CD8+ T-cell specificity can be directly assessed via antigen-specific CD8+ T-cell depletion. The utility of these reagents is demonstrated here in identifying the CD8+ T-cell specificity most effective in preventing HIV progression in HIV-infected HLA-B*27-positive immune controllers.

Adenoviral Vector Vaccination Induces a Conserved Program of CD8(+) T Cell Memory Differentiation in Mouse and Man.

Following exposure to vaccines, antigen-specific CD8(+) T cell responses develop as long-term memory pools. Vaccine strategies based on adenoviral vectors, e.g., those developed for HCV, are able to induce and sustain substantial CD8(+) T cell populations. How such populations evolve following vaccination remains to be defined at a transcriptional level. We addressed the transcriptional regulation of divergent CD8(+) T cell memory pools induced by an adenovector encoding a model antigen (beta-galactosidase). We observe transcriptional profiles that mimic those following infection with persistent pathogens, murine and human cytomegalovirus (CMV). Key transcriptional hallmarks include upregulation of homing receptors and anti-apoptotic pathways, driven by conserved networks of transcription factors, including T-bet. In humans, an adenovirus vaccine induced similar CMV-like phenotypes and transcription factor regulation. These data clarify the core features of CD8(+) T cell memory following vaccination with adenovectors and indicate a conserved pathway for memory development shared with persistent herpesviruses.

CD73 is dispensable for the regulation of inflationary CD8+ T-cells after murine cytomegalovirus infection and adenovirus immunisation.

The ecto-5'-nucleotidase (CD73) is expressed by T-cell subsets, myeloid derived suppressive cells and endothelial cells. It works in conjunction with CD39 to regulate the formation and degradation of adenosine in vivo. Adenosine has previously been shown to suppress the proliferation and cytokine secretion of T-cells and recent evidence suggests that inhibition of CD73 has the potential to enhance T-cell directed therapies. Here we utilised a CD73 knockout mouse model to assess the suppressive ability of CD73 on CD8+ T-cell classical memory and memory "inflation", induced by murine cytomegalovirus (MCMV) infection and adenovirus immunisation. We show that CD73 is dispensable for normal CD8+ T-cell differentiation and function in both models. Thus CD73 as a suppressor of CD8+ T-cells is unlikely to play a deterministic role in the generation and functional characteristics of antiviral memory in these settings.

Autophagy is a critical regulator of memory CD8(+) T cell formation.

During infection, CD8(+) T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8(+) T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8(+) T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8(+) T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice. We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity.

Transient CD8-memory contraction: a potential contributor to latent cytomegalovirus reactivation.

It is clear that latent CMV can reactivate in immunocompetent individuals, but the mechanism triggering such reactivations remains unclear. Recent clinical data suggest that reactivation can be subverted by CMV-specific T-memory. We therefore monitored CMV-specific T cells in immunocompetent mice with latent mCMV after a known reactivation trigger (LPS). LPS induced transient systemic contraction of mCMV-specific CD8 memory that was followed by transcriptional reactivation. Subsequent recovery of mCMV-specific T cells coincided with resumption of latency. These data suggest that bacterial antigen encounters can induce transient T-memory contraction, allowing viral recrudescence in hosts latently infected with herpes family viruses.

A dominant role for the immunoproteasome in CD8+ T cell responses to murine cytomegalovirus.

Murine cytomegalovirus (MCMV) is an important animal model of human cytomegalovirus (HCMV), a ß-Herpesvirus that infects the majority of the world's population and causes disease in neonates and immunocompromised adults. CD8(+) T cells are a major part of the immune response to MCMV and HCMV. Processing of peptides for presentation to CD8(+) T cells may be critically dependent on the immunoproteasome, expression of which is affected by MCMV. However, the overall importance of the immunoproteasome in the generation of immunodominant peptides from MCMV is not known. We therefore examined the role of the immunoproteasome in stimulation of CD8(+) T cell responses to MCMV - both conventional memory responses and those undergoing long-term expansion or "inflation". We infected LMP7(-/-) and C57BL/6 mice with MCMV or with newly-generated recombinant vaccinia viruses (rVVs) encoding the immunodominant MCMV protein M45 in either full-length or epitope-only minigene form. We analysed CD8(+) T cell responses using intracellular cytokine stain (ICS) and MHC Class I tetramer staining for a panel of MCMV-derived epitopes. We showed a critical role for immunoproteasome in MCMV affecting all epitopes studied. Interestingly we found that memory "inflating" epitopes demonstrate reduced immunoproteasome dependence compared to non-inflating epitopes. M45-specific responses induced by rVVs remain immunoproteasome-dependent. These results help to define a critical restriction point for CD8(+) T cell epitopes in natural cytomegalovirus (CMV) infection and potentially in vaccine strategies against this and other viruses.

MHC-peptide tetramers for the analysis of antigen-specific T cells.

The development of the fluorescently labeled tetrameric MHC-peptide complex has enabled the direct visualization, quantification and phenotypic characterization of antigen-specific T cells using flow cytometry and has transformed our understanding of cellular immune responses. The combination of this technology with functional assays provides many new insights into these cells, allowing investigation into their lifecycle, manner of death and effector function. In this article, we hope to provide an overview of the techniques used in the construction of these tetramers, the problems and solutions associated with them, and the methods used in the study of antigen-specific T cells. Understanding how the antigen-specific cells develop and function in different circumstances and with different pathogens will be key to understanding natural host defense, as well as vaccine design and assessment.