Multispecific Response Research Articles

Variability in the impact of linear transportation infrastructures on gene flow in French wild ungulate populations

ContextLinear Transportation Infrastructures (LTIs) are among the largest factors responsible for landscape fragmentation, in turn increasing population isolation. In this context, studies have mainly focused on a single species’ response to barrier elements and mitigation thereof. Yet, the implementation of conservation strategies to restore landscape connectivity may be challenging when multi-specific responses are not measured and fully considered.ObjectivesWe aimed to assess the effect of two different types of LTIs, a fenced highway and a navigation canal on gene flow in three ungulate species in Northeastern France.MethodsWe genotyped 98 red deer (C. elaphus), 120 wild boars (S. scrofa) and 140 roe deer (C. capreolus) with species-specific microsatellite markers from 3 sampling sites located on either side of both LTIs considered in the study area. We assessed the continuity of gene flow using Bayesian clustering methods and a mapping approach to determine inter-individual genetic dissimilarity in relation to landscape characteristics.ResultsOur study showed different impacts of LTIs on the gene flow of species belonging a priori to the same functional group. Genetic differentiation among red deer and wild boar sampling units was observed on either side of the highway, but no such differentiation was identified for roe deer. However, no genetic structuring was associated with the presence of the canal in any species.ConclusionsThe impact of LTIs on gene flow in large species results from the structural characteristics of the infrastructure, and our study shows that mitigation measures should consider species-specific behaviors to facilitate the use of crossing structures and thus ensure gene flow across ILTs.

Can the habitat Ecological Infrastructure’s Diversity Index predict ant and bat biodiversity in Mediterranean agricultural floodplains? A multi-taxon approach using hierarchical modelling

Ecological Infrastructures (EIs) form a network of natural and semi-natural areas crucial to support biodiversity in human-altered landscapes. Several indices have been proposed to assess the ecological quality of EIs, but these have been seldom tested using animal biodiversity as a response variable. The Habitat Ecological Infrastructure's Diversity Index (HEIDI) was recently developed to assess the potential of EIs in supporting biological communities with distinct dispersal abilities. In this study, we evaluated the strength of the HEIDI as a proxy of ant (short-range dispersers) and bat (long-range dispersers) activity in two Mediterranean agricultural areas. For this, we applied a Hierarchical Modelling of Species Communities (HMSC) approach and modelled the multispecific response of ant and bat communities to the HEIDI values and to the scores of the individual categories that compose it. We additionally adopted a functional methodological approach by selecting as response variables the activity of functional guilds, namely animal community regulators, decomposers, and seed dispersers for the ant community, and the activity of echolocation guilds, such as short-range (BAT_SRE), mid-range (BAT_MRE), and long-range echolocators (BAT_LRE) for the bat community. Besides the HEIDI, we also used physiognomic and structural EI attributes, as well as the percentage of the agricultural matrix in the ecological area of influence as covariates in the HMSC approach. Our results showed that the HEIDI formula was particularly accurate in predicting bat activity (posterior mean estimates, βBAT_SRE = 2.80, βBAT_MRE = 0.61, βBAT_LRE = 2.76), but no significant relation was found between HEIDI values and ant activity. In the context of Mediterranean floodplain management, we recommend that practitioners may use the individual HEIDI categorical scores to identify key habitat features that promote ant and bat activity, such as vegetation and aquatic habitats, whilst final HEIDI values may be broadly used to estimate global bat activity.

B cell immunodominance in primary hepatitis C virus infection

Neutralising antibodies (NAbs) play a key role in clearance of HCV. NAbs have been isolated and mapped to several domains on the HCV envelope proteins. However, the immunodominance of these epitopes in HCV infection remains unknown, hindering efforts to elicit optimal epitope-specific responses. Furthermore, it remains unclear which epitope-specific responses are associated with broad NAb (bNAb) activity in primary HCV infection. The aim of this study was to define B cell immunodominance in primary HCV, and its implications on neutralisation breadth and clearance. Using samples from 168 patients with primary HCV infection, the antibody responses targeted 2 immunodominant domains, termed domains B and C. Genotype 1 and 3 infections were associated with responses targeted towards different bNAb domains. No epitopes were uniquely targeted by clearers compared to those who developed chronic infection. Samples with bNAb activity were enriched for multi-specific responses directed towards the epitopes antigenic region 3, antigenic region 4, and domain D, and did not target non-neutralising domains. This study outlines for the first time a clear NAb immunodominance profile in primary HCV infection, and indicates that it is influenced by the infecting virus. It also highlights the need for a vaccination strategy to induce multi-specific responses that do not target non-neutralising domains. Neutralising antibodies will likely form a key component of a protective hepatitis C virus vaccine. In this work we characterise the predominant neutralising and non-neutralising antibody (epitope) targets in acute hepatitis C virus infection. We have defined the natural hierarchy of epitope immunodominance, and demonstrated that viral genotype can impact on this hierarchy. Our findings highlight key epitopes that are associated with broadly neutralising antibodies, and the deleterious impact of mounting a response towards some of these domains on neutralising breadth. These findings should guide future efforts to design immunogens aimed at generating neutralising antibodies with a vaccine candidate.

Epitope characterization of the ADA response directed against a targeted immunocytokine

Targeted immunocytokines (TICs) display potent activity in selective tumor suppression. This class of multi domain biotherapeutics (MDBs) is composed of the three major domains Fab, Fc, and a cytokine which may induce a complex polyclonal anti-drug antibody (ADA) response. However, classical ADA assays usually are not suitable to specify ADAs and to identify the immunogenic domains of a TIC. The purpose of the present study was to establish epitope characterization of ADA responses in order to specify immunogenic responses against a TIC and their direct impact on the pharmacokinetic profile, safety, and efficacy. Based on standard ADA screening and confirmation assays, respectively, domain detection assays (DDAs) and domain competition assays (DCAs) were established and compared by the use of 12 ADA-positive samples obtained from a cynomolgus monkey study in early development. Both domain-specific assays were sensitive enough to preserve the positive screening assay result and revealed an overall accordance for the evaluation of domain-specific ADA responses. About half of the samples displayed one ADA specificity, either for the Fab or for the cytokine (Cy) domain, and the remaining samples showed a combination of Fab-specific and Cy-specific ADA fractions. Fc-specific ADAs occurred in only one sample. In-depth comparison of DCAs and DDAs showed that both assays appeared to be appropriate to assess multi-specific ADA responses as well as minor ADA fractions. An advantage of DCAs is typically a fast and easy assay establishment, whereas, DDAs in some cases may be superior to assess low abundant ADAs in multi-specific responses. Our results reveal that both approaches benefit from thorough reagent development as an essential precondition for reliable epitope characterization of ADA responses.

Hepatitis E virus-specific T-cell response after transplantation

We thank Kamar et al. for their interest in our article. We have to stress that the primary aim of our study was to address basic principles of immune control in hepatitis E virus (HEV) infection and, possibly, to generate additional hypotheses. As in any other study describing, for the first time, an association between a disease condition and a distinct function of immune cells, potential clinical implications obviously need to be confirmed in independent studies. We would therefore be happy to support any other group in testing HEV-specific T-cell responses. Ribavirin, a cheap and easy-to-apply drug, is fortunately effective in most (but not all!) patients with persistent HEV infection. However, ribavirin treatment is not always uncomplicated, because side effects and comorbidities may prevent optimal dosing of ribavirin in single patients. Among several other—maybe more important—findings, one potential novel hypothesis that emerged from our study was that antiviral therapy may be delayed in patients with detectable HEV-specific T-cell responses. The association between strength, frequency, and quality (i.e., interferon-gamma versus interleukin-10) of T-cell responses and HEV RNA status is striking and should be self-explanatory. We are surprised that Kamar et al. 1 neglect the importance of the included control groups in this type of study. Of note, all but 3 HEV RNA-negative transplant patients had acquired HEV after transplantation and thus cleared HEV spontaneously and developed HEV-specific T-cell responses. On the contrary, a correlation between the level of HEV viremia and strength of T-cell responses could not be established because immune responses were very weak or absent in this group. Similar to hepatitis C, we currently do not know the detailed mode of action how ribavirin suppresses HEV replication. Modulation of immune responses could potentially contribute to the clearance of HEV infection. It might indeed be interesting to study, in more detail, the temporal relationship between ribavirin therapy and the evolution of HEV-specific T-cell responses, which was unfortunately not possible here because of limitations in sample availability. We are just in the beginning of understanding potential interactions between HEV and the host's immune system. This topic is of particular interest, because HEV did not evolve as a virus leading to prolonged, or even persisting, infections. The key observations of our study were that we describe, for the first time, the weakness of HEV-specific T-cell responses in chronic hepatitis E, whereas strong, multispecific responses can be observed in resolved patients. The finding that the blocking of certain costimulatory pathways can restore some levels of HEV-specific immunity has implications for understanding immunological mechanisms of chronicity. We invite our colleagues in Toulouse to address similar research questions in their cohort of individuals with chronic HEV infection. However, because the number of chronic hepatitis E patients is still limited, only collaborative projects across centers will be able to answer many of the above-discussed issues. Pothakamuri Venkata Suneetha Ph.D.*, Sven Pischke M.D.*, Heiner Wedemeyer M.D.*, * Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany.

Decline of influenza-specific CD8+ T cell repertoire in healthy geriatric donors

BackgroundWhile influenza vaccination results in protective antibodies against primary infections, clearance of infection is primarily mediated through CD8+ T cells. Studying the CD8+ T cell response to influenza epitopes is crucial in understanding the disease associated morbidity and mortality especially in at risk populations such as the elderly. We compared the CD8+ T cell response to immunodominant and subdominant influenza epitopes in HLA-A2+ control, adult donors, aged 21-42, and in geriatric donors, aged 65 and older.ResultsWe used a novel artificial Antigen Presenting Cell (aAPC) based stimulation assay to reveal responses that could not be detected by enzyme-linked immunosorbent spot (ELISpot). 14 younger control donors and 12 geriatric donors were enrolled in this study. The mean number of influenza-specific subdominant epitopes per control donor detected by ELISpot was only 1.4 while the mean detected by aAPC assay was 3.3 (p = 0.0096). Using the aAPC assay, 92% of the control donors responded to at least one subdominant epitopes, while 71% of control donors responded to more than one subdominant influenza-specific response. 66% of geriatric donors lacked a subdominant influenza-specific response and 33% of geriatric donors responded to only 1 subdominant epitope. The difference in subdominant response between age groups is statistically significant (p = 0.0003).ConclusionGeriatric donors lacked the broad, multi-specific response to subdominant epitopes seen in the control donors. Thus, we conclude that aging leads to a decrease in the subdominant influenza-specific CTL responses which may contribute to the increased morbidity and mortality in older individuals.

Precursor Frequency and Competition Dictate the HLA-A2–Restricted CD8+ T Cell Responses to Influenza A Infection and Vaccination in HLA-A2.1 Transgenic Mice

The human HLA-A2-restricted CD8(+) T cell response to influenza A virus (IAV) is largely directed against the matrix protein-derived M1(58-66) epitope and represents an archetypal example of CD8(+) T cell immunodominance. In this study, we examined the CD8(+) T cell hierarchy to M1(58-66) and two subdominant IAV-specific epitopes: NS1(122-130) and PA(46-55) in HLA-A2(+) human subjects and HLA-A2.1 transgenic (HHD) mice. Using epitope-based lipopeptides, we show that the CD8(+) T cell hierarchy induced by IAV infection could also be induced by lipopeptide vaccination in a context outside of viral infection when the Ag load is equalized. In the HHD HLA-A2.1 mouse model, we show that the naive T cell precursor frequencies, and competition at the Ag presentation level, can predict the IAV-specific CD8(+) T cell hierarchy. Immunization of mice with subdominant epitopes alone was unable to overcome the dominance of the M1(58-66)-specific response in the face of IAV challenge; however, a multiepitope vaccination strategy was most effective at generating a broad and multispecific response to infection.

Decline of influenza-specific T cell repertoire in healthy geriatric donors (85.2)

Abstract While the goal of influenza vaccination is to develop antibodies to protect against a primary infection, clearance of the infection is primarily mediated through CD8+ T cells. The study of the CD8+ T cell response to influenza epitopes is crucial in understanding the disease and associated morbidity and mortality. The age-associated decline of the immune system is believed to be responsible for the increased risk of influenza infection in the geriatric population. Since the CTL response is important for the clearance of influenza infection, we compared the CTL response to immunodominant and subdominant influenza epitopes in HLA-A2+ control adult donors, aged 21-42, to the response in healthy geriatric donors, aged 65 and older. This was done using an artificial Antigen Presenting Cell (aAPC) based stimulation assay which revealed responses that could not be detected by ELISpot. Control donors showed a broad, multi-specific response to the subdominant epitopes, and each donor’s subdominant influenza-specific response was unique. When comparing the control donors’ CTL response to the geriatric donors’, the immunodominant M1 responses were preserved, however, the geriatric donors lacked the broad multi-specific response to influenza-specific subdominant epitopes seen in the control donors. These data indicate that aging leads to a decrease in the subdominant influenza-specific CTL responses which may contribute to the increased morbidity and mortality in older individuals.

Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies.

Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees resulted in robust infection with peak HCV RNA titers of approximately 5.5 log(10) international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necroinflammatory liver changes coinciding with detection of gamma interferon-secreting, intrahepatic T cells. However, the onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multispecific response in the ED43-infected animal (3 weeks before the first evidence of viral control) and a late (week 11) response with limited breadth in the S52-infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes.

Changes in the repertoire of influenza-specific CD8+ T cell responses (130.1)

Abstract Influenza has a high morbidity and mortality rate throughout the world. Geriatric individuals have an increased risk for complications arising from influenza infection and account for over 90% of influenza related deaths. To assess why geriatrics are unable to combat influenza infection as well as adult individuals, we used artificial antigen presenting cells (aAPC) to generate influenza-specific CD8+ T cells ex vivo from healthy donors aged 21-60 and geriatric donors over the age of 65. We found a very diverse response to the subdominant influenza peptides, PB1413-421, NS1123-132, NA231-239, NA75-84, PA46-54, and PA225-233 in the adult donors. While we were able to generate CD8+ T cells against the immunodominant influenza M158-66 peptide in all donors, the geriatric donors lacked the broad, multi-specific response to the subdominant influenza epitopes that we found in the adult donors. These results suggest that aging leads to a hole in the T cell receptor (TCR) repertoire and the presence of CD8+ T cells against subdominant influenza peptides plays a major role in clearance of influenza infection.

Multispecific responses by T cells expanded by endogenous self‐peptide/MHC complexes

The paradox of autoreactivity to self-peptides in physiological as opposed to pathological immune responses is not well understood. Here, we directly examined the human T cell response to endogenous self-peptides in a series of healthy subjects. CFSE-labeled T cells were stimulated with unmanipulated antigen-presenting cells containing endogenous self-antigen, and the resulting CD4+ populations entering into cell cycle (CFSE(low)) or non-proliferating CD4+ cells (CFSE(high)) were single-cell sorted, cloned and screened against a panel of self-antigens and microbial recall antigens to interrogate their antigen reactivity. The percentage of CD4+ T cells entering cell cycle in response to self-peptide/MHC was calculated to be 0.04%, and entry into cell cycle was dependent upon CD28 costimulation. Clones derived from CFSE(low) T cells exhibited significantly greater cross-reactivity to multiple antigens than CFSE(high) clones or other CD4+ clones generated after microbial antigen stimulation. Sequencing the TCRbeta chains indicated that CFSE(low) clones were indeed clonal. These data demonstrate that T cell clones generated on stimulation by endogenous self-peptides exhibit a high degree of multispecificity, and we speculate that their multispecificity is based upon recognition of shared-backbone MHC determinants.

Subversion of effector CD8+ T cell differentiation in acute hepatitis C virus infection: exploring the immunological mechanisms.

Hallmark of acute hepatitis C virus (HCV) infection is a severe virus-specific effector CD8(+) T cell dysfunction that seems to be a critical factor in preventing the resolution of infection and in favoring the onset of chronic liver immunopathology. We suggest that this dysfunction is critical in the establishment of HCV persistence, unless it is compensated by multispecific responses, as found in individuals resolving infection. Analyses on purified populations indicate that central memory HCV-specific CCR7(+)/CD8(+) T cells efficiently proliferate and differentiate in vitro, although the large population of memory effector CCR7(-) cells found in the peripheral blood of acutely infected patients display poor effector functions ex vivo (semi-effectors). However, we report strong evidence in support of IL-2 being capable of pushing semi-effector CTL to complete their effector cell program. Therefore, IL-2 deficiency during T cell activation may be responsible for the dichotomy between memory CTL expansion and incomplete effector differentiation shown in patients with acute HCV infection. These data are consistent with the possible therapeutic treatment with IL-2 to rebuild the effector T cell pool in these patients.

Comprehensive screening reveals strong and broadly directed human immunodeficiency virus type 1-specific CD8 responses in perinatally infected children.

Advances in antiviral therapy have dramatically shifted the demographics of pediatric human immunodeficiency virus type 1 (HIV-1) infection in the developed world, and a growing proportion of perinatally HIV-1-infected children are now entering their second or even third decade of life. Although cellular immune responses to HIV are known to be weak in early infancy, the magnitude, breadth, and specificity of responses later in childhood have not been characterized in detail. We performed a comprehensive characterization of HIV-1-specific CD8 responses in 18 perinatally infected children (age range, 6 to 17 years), most of whom were on antiviral therapy, using both previously defined HIV-1 epitopes and overlapping peptides spanning all HIV-1 proteins. Multispecific responses were detected in all subjects and accounted for a median of 0.25 to 0.3% of all peripheral blood mononuclear cells that was similar to the magnitude seen in HIV-infected adults. CD8 responses were broadly directed at an average of 11 epitopes (range, 2 to 27 epitopes) and targeted nearly all HIV-1 proteins, with the highest proportion in Gag. Responses were readily detected even in those children with suppressed viremia on highly active antiretroviral therapy, although the breadth (P = 0.037) and the magnitude (P = 0.021) were significantly lower in these subjects. Each child recognized only a small minority of the HIV-1 optimal epitopes defined for his or her class I HLA alleles. Together, these data indicate that perinatally infected children who survive infancy mount a robust HIV-1-specific CD8 response that is much stronger than previously thought and is comparable in magnitude and breadth to that of adults. Moreover, this response has the potential to be broadened to target more epitopes, making these children attractive candidates for immunotherapeutic interventions.

A rational strategy to design multiepitope immunogens based on multiple Th lymphocyte epitopes.

Four HLA-DR-restricted HIV-derived Th lymphocyte (HTL) epitopes cross-reactive with the murine I-A(b) class II molecule were used to evaluate different vaccine design strategies to simultaneously induce multiple HTL responses. All four epitopes were immunogenic in H-2(b) mice, demonstrating the feasibility of murine models to evaluate epitope-based vaccines destined for human use. Immunization with a pool of peptides induced responses against all four epitopes; illustrating immunodominance does not prevent the induction of balanced multispecific responses. When different delivery systems were evaluated, a multiple Ag peptide construct was found to be less efficient than a linear polypeptide encompassing all four epitopes. Further characterization of linear polypeptide revealed that the sequential arrangement of the epitopes created a junctional epitope with high affinity class II binding. Disruption of this junctional epitope through the introduction of a GPGPG spacer restored the immunogenicity against all four epitopes. Finally, we demonstrate that a GPGPG spacer construct can be used to induce HTL responses by either polypeptide or DNA immunization, highlighting the flexibility of the approach.

Chemoselective Ligation and Antigen Vectorization

The interest in cocktail-lipopeptide vaccines has now been confirmed by phase I clinical trials: highly diversified B-, T-helper or cytotoxic T-cell epitopes can be combined with a lipophilic vector for the induction of B- and T-cell responses of predetermined specificity. With the goal of producing an improved vaccine that should ideally induce a multispecific response in non-selected populations, increasing the diversity of the immunizing mixture represents one of the most obvious strategies.The selective delivery of antigens to professional antigen-presenting cells represents another promising approach for the improvement of vaccine efficacy. In this context, the mannose-receptor represents an attractive entry point for the targeting to dendritic cells of antigens linked to clustered glycosides or glycomimetics.In all cases, highly complex but fully characterized molecules must be produced. To develop a modular and flexible strategy which could be generally applicable to a large set of peptide antigens, we elected to explore the potentialities of chemoselective ligation methods. The hydrazone bond was found particularly reliable and fully compatible with sulphide ligation. Hydrazone/thioether orthogonal ligation systems could be developed to account for the nature of the antigens and the solubility of the vector systems.

Donor heart endothelial cells as targets for graft infiltrating lymphocytes after clinical cardiac transplantation

The in vitro cytotoxic reactivity of allograft infiltrating cells cultured from endomyocardial biopsies was tested against endothelial cells (EC) isolated from the own donor heart. EC derived from pieces of atrium were found to be proper targets for graft infiltrating cytotoxic T cells from four patients. The specificity of this cytotoxicity was further analysed by cold target inhibition studies and blocking with anti-CD3, anti-CD4 or anti-CD8 monoclonal antibodies. The experiments revealed that, besides a clearly HLA directed recognition, a more heterogeneous, multispecific response can be found, which might be partially explained by the activity of EC specific T cell clones. We conclude that this system provides a valuable approach to investigate the reactivity of graft infiltrating cells against EC in relation to the clinical course of the transplantation.

Monoclonal xenogeneic antibodies to mouse leukocyte antigens: identification of macrophage-specific and other differentiation antigens.

The identification and study of cell surface molecules which have specific immune functions and are markers of differentiated white blood cell subpopulations is of great interest in immunology. Antibodies are versatile probes with which to study these molecules. However, it is difficult to obtain highly specific antibodies recognizing individual cell surface molecules, because cell surfaces are complex mosaics of many different immunogenic glycoproteins and glycolipids. A general approach to this problem stems from the experiments of Köhler and Milstein (1). They fused myeloma cells and spleen cells from mice immunized with sheep red blood cells to derive continuous hybrid cell lines secreting antibodies to sheep red blood cells. Such lines can be manipulated in culture so that the multispecific response to a complex immunogen can be resolved into a set of monospecific responses by cloning. Hybrids have been obtained which secrete antibodies to rat major histocompatibility antigens(2), rat cell surface xenoantigens (3), mouse IgD allotypes (4), and mouse H-2K antigens (5).KeywordsSpleen CellAntibody Secreting CellAntigen TiterMajor Histocompatibility AntigenCell Surface Differentiation AntigenThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.