Search

Streptococcus pneumoniae (S. pneumoniae) infections are the leading cause of child mortality globally. Current vaccines fail to induce a protective immune response towards a conserved part of the pathogen, resulting in new serotypes causing disease. Therefore, new vaccine strategies are urgently needed. Described is a two-pronged approach combining S. pneumoniae proteins, pneumolysin (Ply) and pneumococcal surface protein A (PspA), with a precisely defined synthetic oligosaccharide, whereby the carrier protein acts as a serotype-independent antigen to provide additional protection. Proof of concept in mice and swine models revealed that the conjugates inhibited colonization of the nasopharynx, decreased the bacterial load and reduced disease severity in the bacteria challenge model. Immunization of piglets provided the first evidence for the immunogenicity and protective potential of synthetic glycoconjugate vaccine in a large animal model. A combination of synthetic oligosaccharides with proteins from the target pathogen opens the path to create broadly cross-protective (“universal”) pneumococcal vaccines.

Seasonal Influenza A virus (IAV) infections can promote dissemination of upper respiratory tract commensals such asStreptococcus pneumoniaeto the lower respiratory tract resulting in severe life-threatening pneumonia. Here, we aimed to compare innate immune responses in the lungs of healthy colonized and non-colonized mice after IAV challenge at the initial asymptomatic stage of infection. Responses during a severe bacterial pneumonia were profiled for comparison. Cytokine and innate immune cell imprints of the lungs were analyzed. Irrespective of the colonization status, mild H1N1 IAV infection was characterized by a bi-phasic disease progression resulting in full recovery of the animals. Already at the asymptomatic stage of viral infection, the pro-inflammatory cytokine response was as high as in pneumococcal pneumonia. Flow cytometry analyses revealed an early influx of inflammatory monocytes into the lungs. Neutrophil influx was mostly limited to bacterial infections. The majority of cells, except monocytes, displayed an activated phenotype characterized by elevated CCR2 and MHCII expression. In conclusion, we show that IAV challenge of colonized healthy mice does not automatically result in severe co-infection. However, a general local inflammatory response was noted at the asymptomatic stage of infection irrespective of the infection type.

Infection with African swine fever virus (ASFV) causes a highly lethal haemorrhagic disease in domestic and Eurasian wild pigs. Thus, it is a major threat to pig populations worldwide and a cause of substantial economic losses. Recently, less virulent ASFV strains emerged naturally, which showed higher experimental virulence in wild boar than in domestic pigs. The reason for this difference in disease progression and outcome is unclear but likely involves different immunological responses. Unfortunately, besides the importance of CD8α+ lymphocytes, little is known about the immune responses against ASFV in suids. Against this background, we used a multicolour flow cytometry platform to investigate the T-cell responses in wild boar and domestic pigs after infection with the moderately virulent ASFV strain 'Estonia2014' in two independent trials. CD4- /CD8α+ and CD4+ /CD8α+ αβ T-cell frequencies increased in both subspecies in various tissues, but CD8α+ γδ T cells differentiated and responded in wild boar only. Proliferation in CD8α+ T cells was found 10 days post infectionem only. Frequencies of T-bet+ T cells increased in wild boar but not in domestic pigs. Of note, we found a considerable loss of perforin expression in cytotoxic T cells, 5 and 7 dpi. Both subspecies established a regulatory T-cell response 10 dpi. In domestic pigs, we show increasing levels of ICOS+ and CD8α+ invariant Natural Killer T cells. These disparities in T-cell responses might explain some of the differences in disease progression in wild boar and domestic pigs and should pave the way for future studies.

Since African swine fever (ASF) first appeared in the Caucasus region in 2007, it has spread rapidly and is now present in numerous European and Asian countries. In Europe, mainly wild boar populations are affected and pose a risk for domestic pigs. In Asia, domestic pigs are almost exclusively affected. An effective and safe vaccine is not available, and correlates of protection are far from being understood. Therefore, research on immune responses, immune dysfunction, and pathogenesis is mandatory. It is acknowledged that T cells play a pivotal role. Thus, we investigated T cell responses of domestic pigs and wild boar upon infection with the highly virulent ASF virus (ASFV) strain “Armenia08”. For this purpose, we used a flow cytometry‐based multicolor analysis to identify T cell subtypes (cytotoxic T cells, T‐helper cells, γʂ T cells) and their functional impairment in ASFV infected pigs. Domestic pigs showed lymphopenia and neither in the blood nor in the lymphoid organs was a proliferation of CD8+ effector cells observed. Furthermore, a T‐bet‐dependent activation of the remaining CD8 T cells did not occur. In contrast, a T cell response could be observed in wild boar at 5 days post inoculation in the blood and in tendency also in some organs. However, this cytotoxic response was not beneficial as all wild boars showed a severe acute lethal disease and a higher proportion died spontaneously or was euthanized at the humane endpoint.

African swine fever virus (ASFV), causing an OIE-notifiable viral disease of swine, is spreading over the Eurasian continent and threatening the global pig industry. Here, we conducted the first proteome analysis of ASFV-infected primary porcine monocyte-derived macrophages (moMΦ). In parallel to moMΦ isolated from different pigs, the stable porcine cell line WSL-R was infected with a recombinant of ASFV genotype IX strain “Kenya1033”. The outcome of the infections was compared via quantitative mass spectrometry (MS)-based proteome analysis. Major differences with respect to the expression of viral proteins or the host cell response were not observed. However, cell-specific expression of some individual viral proteins did occur. The observed modulations of the host proteome were mainly related to cell characteristics and function. Overall, we conclude that both infection models are suitable for use in the study of ASFV infection in vitro.

Herpes simplex encephalitis (HSE) is one of the most serious diseases of the nervous system in humans. However, its pathogenesis is still only poorly understood. Although several mouse models of predominantly herpes simplex virus 1 (HSV-1) infections mimic different crucial aspects of HSE, central questions remain unanswered. They comprise the specific temporofrontal tropism, viral spread within the central nervous system (CNS), as well as potential molecular and immunological barriers that drive virus into latency while only rarely resulting in severe HSE. We have recently proposed an alternative mouse model by using a pseudorabies virus (PrV) mutant that more faithfully represents the striking features of human HSE: temporofrontal meningoencephalitis with few severely, but generally only moderately to subclinically affected mice as well as characteristic behavioral abnormalities. Here, we characterized this animal model using 6- to 8-week-old female CD-1 mice in more detail. Long-term investigation over 6 months consistently revealed a biphasic course of infection accompanied by recurring clinical signs including behavioral alterations and mainly mild meningoencephalitis restricted to the temporal and frontal lobes. By histopathological and immunological analyses, we followed the kinetics and spatial distribution of inflammatory lesions as well as the underlying cytokine expression in the CNS over 21 days within the acute phase of infection. Affecting the temporal lobes, the inflammatory infiltrate was composed of lymphocytes and macrophages showing a predominantly lymphocytic shift 15 days after infection. A strong increase was observed in cytokines CXCL10, CCL2, CCL5, and CXCL1 recruiting inflammatory cells to the CNS. Unlike the majority of infected mice, strongly affected animals demonstrated extensive temporal lobe edema, which is typically present in severe human HSE cases. In summary, these results support the validity of our animal model for in-depth investigation of HSE pathogenesis.

Avian influenza viruses (AIV) H5N8 clade 2.3.4.4 pose a public health threat but the viral factors relevant for its potential adaptation to mammals are largely unknown. The non-structural protein 1 (NS1) of influenza viruses is an essential interferon antagonist. It commonly consists of 230 amino acids, but variations in the disordered C-terminus resulted in truncation or extension of NS1 with a possible impact on virus fitness in mammals. Here, we analysed NS1 sequences from 1902 to 2020 representing human influenza viruses (hIAV) as well as AIV in birds, humans and other mammals and with an emphasis on the panzootic AIV subtype H5N8 clade 2.3.4.4A (H5N8-A) from 2013 to 2015 and clade 2.3.4.4B (H5N8-B) since 2016. We found a high degree of prevalence for short NS1 sequences among hIAV, zoonotic AIV and H5N8-B, while AIV and H5N8-A had longer NS1 sequences. We assessed the fitness of recombinant H5N8-A and H5N8-B viruses carrying NS1 proteins with different lengths in human cells and in mice. H5N8-B with a short NS1, similar to hIAV or AIV from a human or other mammal-origins, was more efficient at blocking apoptosis and interferon-induction without a significant impact on virus replication in human cells. In mice, shortening of the NS1 of H5N8-A increased virus virulence, while the extension of NS1 of H5N8-B reduced virus virulence and replication. Taken together, we have described the biological impact of variation in the NS1 C-terminus in hIAV and AIV and shown that this affects virus fitness in vitro and in vivo.

Streptococcus pneumoniaeinfections are the leading cause of child mortality globally. Current vaccines fail to induce a protective immune response towards a conserved part of the pathogen, resulting in new serotypes causing disease. Therefore, new vaccine strategies are urgently needed. Described is a two-pronged approach combiningS.pneumoniaeproteins, pneumolysin and PspA, with a precisely defined synthetic oligosaccharide, whereby the carrier protein acts as a serotype-independent antigen to provide additional protection. Proof of concept in mice and swine models revealed that the conjugates inhibit colonization of the nasopharynx, decrease the bacterial load and reduce disease severity in the bacteria challenged model. Immunization of piglets provided the first evidence for the immunogenicity and protective potential of synthetic glycoconjugate vaccine in a large animal model. A combination of synthetic oligosaccharides with proteins from the target pathogen opens the path to create broadly cross-protective (“universal“) pneumococcal vaccines.

Influenza A viruses (IAV) have caused seasonal epidemics and severe pandemics in humans. Novel pandemic strains as in 2009 may emerge from pigs, serving as perpetual virus reservoir. However, reliably effective vaccination has remained a key issue for humans and swine. Here, we generated a novel double-attenuated influenza live vaccine by reverse genetics and subjected immunized mice and pigs to infection with the homologous wild-type, another homosubtypic H1N1, or a heterosubtypic H3N2 virus to address realistic challenge constellations. This attenuated mutant contains an artificial, strictly elastase-dependent hemagglutinin cleavage site and a C-terminally truncated NS1 protein from the IAV A/Bayern/74/2009 (H1N1pdm09). Prior to challenge, we immunized mice once and pigs twice intranasally. In vitro, the double-attenuated mutant replicated strictly elastase-dependently. Immunized mice and pigs developed neither clinical symptoms nor detectable virus replication after homologous challenge. In pigs, we observed considerably reduced clinical signs and no nasal virus shedding after homosubtypic and reduced viral loads in respiratory tracts after heterosubtypic infection. Protection against homosubtypic challenge suggests that an optimized backbone strain may require less frequent updates with recent HA and NA genes and still induce robust protection in relevant IAV hosts against drifted viruses.

The unprecedented spread of H5N8- and H9N2-subtype avian influenza virus (AIV) in birds across Asia, Europe, Africa, and North America poses a serious public health threat with a permanent risk of reassortment and the possible emergence of novel virus variants with high virulence in mammals. To gain information on this risk, we studied the potential for reassortment between two contemporary H9N2 and H5N8 viruses. While the replacement of the PB2, PA, and NS genes of highly pathogenic H5N8 by homologous segments from H9N2 produced infectious H5N8 progeny, PB1 and NP of H9N2 were not able to replace the respective segments from H5N8 due to residues outside the packaging region. Furthermore, exchange of the PB2, PA, and NS segments of H5N8 by those of H9N2 increased replication, polymerase activity and interferon antagonism of the H5N8 reassortants in human cells. Notably, H5N8 reassortants carrying the H9N2-subtype PB2 segment and to lesser extent the PA or NS segments showed remarkably increased virulence in mice as indicated by rapid onset of mortality, reduced mean time to death and increased body weight loss. Simultaneously, we observed that in chickens the H5N8 reassortants, particularly with the H9N2 NS segment, demonstrated significantly reduced transmission to co-housed chickens. Together, while the limited capacity for reassortment between co-circulating H9N2 and H5N8 viruses and the reduced bird-to-bird transmission of possible H5N8 reassortants in chickens may limit the evolution of such reassortant viruses, they show a higher replication potential in human cells and increased virulence in mammals.

Endemically infected European wild boar are considered a major reservoir of African swine fever virus in Europe. While high lethality was observed in the majority of field cases, strains of moderate virulence occurred in the Baltic States. One of these, &ldquo, Estonia 2014&rdquo, led to a higher number of clinically healthy, antibody-positive animals in the hunting bag of North-Eastern Estonia. Experimental characterization showed high virulence in wild boar but moderate virulence in domestic pigs. Putative pathogenic differences between wild boar and domestic pigs are unresolved and comparative pathological studies are limited. We here report on a kinetic experiment in both subspecies. Three animals each were euthanized at 4, 7, and 10 days post infection (dpi). Clinical data confirmed higher virulence in wild boar although macroscopy and viral genome load in blood and tissues were comparable in both subspecies. The percentage of viral antigen positive myeloid cells tested by flow cytometry did not differ significantly in most tissues. Only immunohistochemistry revealed consistently higher viral antigen loads in wild boar tissues in particular 7 dpi, whereas domestic pigs already eliminated the virus. The moderate virulence in domestic pigs could be explained by a more effective viral clearance.

The introduction of genotype II African swine fever (ASF) virus, presumably from Africa into Georgia in 2007, and its continuous spread through Europe and Asia as a panzootic disease of suids, continues to have a huge socio-economic impact. ASF is characterized by hemorrhagic fever leading to a high case/fatality ratio in pigs. In Europe, wild boar are especially affected. This review summarizes the currently available knowledge on ASF in wild boar in Europe. The current ASF panzootic is characterized by self-sustaining cycles of infection in the wild boar population. Spill-over and spill-back events occur from wild boar to domestic pigs and vice versa. The social structure of wild boar populations and the spatial behavior of the animals, a variety of ASF virus (ASFV) transmission mechanisms and persistence in the environment complicate the modeling of the disease. Control measures focus on the detection and removal of wild boar carcasses, in which ASFV can remain infectious for months. Further measures include the reduction in wild boar density and the limitation of wild boar movements through fences. Using these measures, the Czech Republic and Belgium succeeded in eliminating ASF in their territories, while the disease spread in others. So far, no vaccine is available to protect wild boar or domestic pigs reliably against ASF.

Pigs are anatomically, genetically and physiologically comparable to humans and represent a natural host for influenza A virus (IAV) infections. Thus, pigs may represent a relevant biomedical model for human IAV infections. We set out to investigate the systemic as well as the local immune response in pigs upon two subsequent intranasal infections with IAV H1N1pdm09. We detected decreasing numbers of peripheral blood lymphocytes after the first infection. The simultaneous increase in the frequencies of proliferating cells correlated with an increase in infiltrating leukocytes in the lung. Enhanced perforin expression in αβ and γδ T cells in the respiratory tract indicated a cytotoxic T cell response restricted to the route of virus entry such as the nose, the lung and the bronchoalveolar lavage. Simultaneously, increasing frequencies of CD8αα expressing αβ T cells were observed rapidly after the first infection, which may have inhibited uncontrolled inflammation in the respiratory tract. Taking together, the results of this study demonstrate that experimental IAV infection in pigs mimics major characteristics of human seasonal IAV infections.

Pigs are important livestock and comprehensive understanding of their immune responses in infections is critical to improve vaccines and therapies. Moreover, similarities between human and swine physiology suggest that pigs are a superior animal model for immunological studies. However, paucity of experimental tools for a systematic analysis of the immune responses in pigs represent a major disadvantage. To evaluate the pig as a biomedical model and additionally expand the knowledge of rare immune cell populations in swine, we established a multicolor flow cytometry analysis platform of surface marker expression and cellular responses for porcine invariant Natural Killer T cells (iNKT). In humans, iNKT cells are among the first line defenders in various tissues, respond to CD1d-restricted antigens and become rapidly activated. Naïve porcine iNKT cells were CD3+/CD4−/CD8+ or CD3+/CD4−/CD8− and displayed an effector- or memory-like phenotype (CD25+/ICOS+/CD5hi/CD45RA−/CCR7 ± /CD27+). Based on their expression of the transcription factors T bet and the iNKT cell-specific promyelocytic leukemia zinc finger protein (PLZF), porcine iNKT cells were differentiated into functional subsets. Analogous to human iNKT cells, in vitro stimulation of porcine leukocytes with the CD1d ligand α-galactosylceramide resulted in rapid iNKT cell proliferation, evidenced by an increase in frequency and Ki-67 expression. Moreover, this approach revealed CD25, CD5, ICOS, and the major histocompatibility complex class II (MHC II) as activation markers on porcine iNKT cells. Activated iNKT cells also expressed interferon-γ, upregulated perforin expression, and displayed degranulation. In steady state, iNKT cell frequency was highest in newborn piglets and decreased with age. Upon infection with two viruses of high relevance to swine and humans, iNKT cells expanded. Animals infected with African swine fever virus displayed an increase of iNKT cell frequency in peripheral blood, regional lymph nodes, and lungs. During Influenza A virus infection, iNKT cell percentage increased in blood, lung lymph nodes, and broncho-alveolar lavage. Our in-depth characterization of porcine iNKT cells contributes to a better understanding of porcine immune responses, thereby facilitating the design of innovative interventions against infectious diseases. Moreover, we provide new evidence that endorses the suitability of the pig as a biomedical model for iNKT cell research.

To evaluate the effect of selection for high laying performance on the capacity to respond to an infection with avian influenza virus (AIV), four different chicken lines were tested: A white layer and a brown layer breed originating from a commercial breeding program, and a white layer and a brown layer line maintained as a conservation flock for decades without any selection. The different chicken breeds were infected with AIV of different pathotypes (low pathogenic to high pathogenic) to evaluate and compare their immunological competence. Morbidity and mortality rates, as well as viral shedding, were investigated as parameters of virus infection. Immune cells in blood samples collected after different time points following inoculation were identified. In general, the chickens of the two phylogenetically related brown layer lines (irrespective of the performance type) were more resistant to infection with the selected AIVs, reflected by a lower mortality rate (low virulent AIV) or a prolonged length of survival before succumbing to the disease (highly virulent AIV). Corresponding to these results, CD8-positive cell counts were reduced in both white layer lines. This observation was also confirmed in an in vivo allogenic transfer experiment, in which brown layers eliminated the transferred cells in a shorter time period. In conclusion, our results do not support the theory of reduced immunological competence of high-performance layer breeds, at least against AIV infection. Instead, brown layer strains had a faster CD8-positive immune cell response after viral or allogenic stimulus than the phylogenetically distant white layers, resulting in better resistance against AIV infection.

The development of immunocompetence in chicks after hatching is not fully understood. However, detailed knowledge of immunocompetence and maturation processes in day-old chicks (DOCs) and juvenile chickens (Gallus gallus domesticus) is necessary to implement enhanced immunization strategies. For viral diseases, this especially includes the development of cellular immunity focusing on T-cell-dependent responses. In the current study, we investigated T-cell subsets in blood and lymphoid tissues of 1-to-21-day-old chickens concerning their cellular composition and localization. We detected an increase of T-cell frequencies in blood and spleen and a shift of the CD8α dimer expression toward a CD8αβ expression on the surface of T cells with increasing age. A relocalization of lymphocytes into antigen presentation structures within the spleen was affirmed. In addition, changes in basal messenger RNA (mRNA) level, with increasing IL2 and IFNγ mRNA levels at different ages were measured. These detected changes suggest an improved T-cell-dependent antiviral response with increasing age in chickens. To confirm this finding on a functional level, we conducted a transfer experiment: adult and, as a negative control, neonatal naive lymphocytes were transferred into DOCs. Afterward, the protection induced by these transferred cells was verified by a sublethal infection by using a highly pathogenic avian influenza virus with neuraminidase deletion, H5Ndel. Previous experiments have shown that adult animals survive infection with this virus strain, while naive DOCs show severe symptoms or even die. As a result, the transfer of adult, but not neonatal lymphocytes, confers protection to DOCs against the infection, demonstrating functional differences in lymphocytes from chicks of different ages. Collectively, these data reveal the inability of chicks to mount an effective, cellular antiviral response in the first 3 wk of life. Therefore, we propose that the observed maturation of both the innate and the adaptive arms of the immune system early in development is mandatory for controlling influenza infection in chickens, as well as for an effective vaccination with replication-competent viral vaccine strains.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell-skewing vaccine adjuvants.

Influenza is an important respiratory disease of pigs, and may lead to novel human pathogens like the 2009 pandemic H1N1 swine-origin influenza virus (SoIV). Therefore, improved influenza vaccines for pigs are required. Recently, we demonstrated that single intranasal immunization with a hemagglutinin (HA)-expressing pseudorabies virus recombinant of vaccine strain Bartha (PrV-Ba) protected pigs from H1N1 SoIV challenge (Klingbeil et al., 2014). Now we investigated enhancement of efficacy by prime-boost vaccination and/or intramuscular administration. Furthermore, a novel PrV-Ba recombinant expressing codon-optimized N1 neuraminidase (NA) was included. In vitro replication of this virus was only slightly affected compared to parental virus. Unlike HA, the abundantly expressed NA was efficiently incorporated into PrV particles. Immunization of pigs with the two PrV recombinants, either singly or in combination, induced B cell proliferation and the expected SoIV-specific antibodies, whose titers increased substantially after boost vaccination. After immunization of animals with either PrV recombinant H1N1 SoIV challenge virus replication was significantly reduced compared to PrV-Ba vaccinated or naïve controls. Protective efficacy of HA-expressing PrV was higher than of NA-expressing PrV, and not significantly enhanced by combination. Despite higher serum antibody titers obtained after intramuscular immunization, transmission of challenge virus to naïve contact animals was only prevented after intranasal prime-boost vaccination with HA-expressing PrV-Ba.

Pancreatic cancer is the 8th most common cause of cancer-related deaths worldwide and the tumor with the poorest prognosis of all solid malignancies. In 1957, it was discovered that Newcastle disease virus (NDV) has oncolytic properties on tumor cells. To study the oncolytic properties of NDV in pancreatic cancer a single dose was administered intravenously in a syngeneic orthotopic tumor model using two different murine pancreatic adenocarcinoma cell lines (DT6606PDA, Panc02). Tumor growth was monitored and immune response was analyzed. A single treatment with NDV inhibited DT6606PDA tumor growth in mice and prevented recurrence for a period of three months. Tumor infiltration and systemic activation of NK cells, cytotoxic and helper T-cells was enhanced. NDV-induced melting of Panc02 tumors until d7 pi, but they recurred displaying unrestricted tumor growth, low immunogenicity and inhibition of tumor-specific immune response. Arrest of DT6606PDA tumor growth and rejection was mediated by activation of NK cells and a specific antitumor immune response via T-cells. Panc02 tumors rapidly decreased until d7 pi, but henceforth tumors characterized by the ability to perform immune-regulatory functions reappeared. Our results demonstrated that NDV-activated immune cells are able to reject tumors provided that an adaptive antitumor immune response can be initiated. However, activated NK cells that are abundant in Panc02 tumors lead to outgrowth of nonimmunogenic tumor cells with inhibitory properties. Our study emphasizes the importance of an adaptive immune response, which is initiated by NDV to mediate long-term tumor surveillance in addition to direct oncolysis.

Nipah virus (NiV), a BSL-4 pathogen, belongs to the genus Henipavirus within the family Paramyxoviridae. To date, no effective vaccine is available. Although most of the current vaccine studies aim to induce a neutralizing antibody response, it has become evident that a promising vaccine should target both, humoral and cell-mediated immune response. Virus-like particles (VLPs) have been shown to activate both arms of the adaptive immune response. In our study, VLPs composed of the NiV surface glycoproteins G and F and the matrix protein of the closely related Hendra virus (HeV M) induced both, a neutralizing antibody response and an antigen-specific CD8 T cell response with proliferation, IFN-γ expression and Th1 cytokine secretion in C57BL/6 mice. In contrast, in BALB/c mice only a neutralizing antibody response was observed. All three viral proteins included in the VLPs were shown to harbor CD8 T cell epitopes; however, the combination of all three proteins enhanced the magnitude of the CD8 T cell response. To conclude, VLPs represent a promising vaccine candidate, as they induce humoral as well as CD8 T cell-mediated immune responses.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (T

Influenza virus A/H1N1, which is currently causing a pandemic, contains gene segments with ancestors in the North American and Eurasian swine lineages. To get insights into virus replication dynamics, clinical symptoms and virus transmission in pigs, we infected animals intranasally with influenza virus A/Regensburg/D6/09/H1N1. Virus excretion in the inoculated pigs was detected in nasal swabs from 1 day post-infection (p.i.) onwards and the pigs developed generally mild symptoms, including fever, sneezing, nasal discharge and diarrhoea. Contact pigs became infected, shed virus and developed clinical symptoms similar to those in the inoculated animals. Plasma samples of all animals remained negative for virus RNA. Nucleoprotein- and haemagglutinin H1-specific antibodies could be detected by ELISA 7 days p.i. CD4(+) T cells became activated immediately after infection and both CD4(+) and CD8(+) T-cell populations expanded from 3 to 7 days p.i., coinciding with clinical signs. Contact chickens remained uninfected, as judged by the absence of virus excretion, clinical signs and seroconversion.

Two secreted proteins were detected in culture supernatants of PHA or PMA stimulated, immunomagnetically separated, sIgM − leucocytes of rainbow trout ( Oncorhynchus mykiss ) with 60 kDa and with 12–15 kDa (multiple bands). So called conditioned media (CM), containing these proteins, induced significant activation of blood and head kidney leucocytes. Immunomagnetically separated, naive as well as PHA activated sIgM − T lymphocytes and LPS prestimulated sIgM + B lymphocytes could be identified to be responding to these secreted proteins. Using a monoclonal antibody specific for mouse IL-2 (clone JES6-1A12), one of the multiple 12–15 kDa proteins could be stained in Western blots. It was also shown that the induced proliferation was due to this protein in the CM, as the same anti-IL-2 mab was able to block the CM induced proliferation. Furthermore, survival of the IL-2 dependent mouse cell line HT-2 was enhanced after addition of various concentrations of CM. The data presented show, for the first time, that mitogen stimulated trout sIgM − leucocytes secrete a cytokine like growth factor sharing functional and structural similarities with mammalian IL-2.

Tumor-specific CD8 T cell responses to MCA102 fibrosarcoma cells expressing the cytotoxic T cell epitope gp33 from lymphocytic choriomeningitis virus were studied. MCA102gp33 tumors grew progressively in C57BL/6 mice, despite induction of peripheral gp33-tetramer+ T cells that were capable of mediating antiviral protection, specific cell rejection, and concomitant tumor immunity. MCA102gp33 tumors were infiltrated with a high number (∼20%) of CD11b+CD11c− macrophage-phenotype cells that were able to cross-present the gp33 epitope to T cells. Tumor-infiltrating CD8 T cells exhibited a highly activated phenotype but lacked effector cell function. Strikingly, a significant portion of tumor-infiltrating lymphocytes expressed TCRs specific for gp33 but bound MHC tetramers only after cell purification and a 24-h resting period in vitro. The phenomenon of “tetramer-negative T cells” was not restricted to tumor-infiltrating lymphocytes from MCA102gp33 tumors, but was also observed when Ag-specific T cells derived from an environment with high Ag load were analyzed ex vivo. Thus, using a novel tumor model, allowing us to trace tumor-specific T cells at the single cell level in vivo, we demonstrate that the tumor microenvironment is able to alter the functional activity of T cells infiltrating the tumor mass.

Background Classical swine fever (CSF) is one of the most important viral diseases of pigs. Clinical signs may vary from almost inapparent infection to a hemorrhagic fever like illness. Among the host factors leading to different disease courses are age, breed, and immune status. The aim of this study was to compare host responses of different pig breeds upon infection with a recent moderately virulent CSF virus (CSFV) strain, and to assess their impact on the clinical outcome and the efficiency of immune responses. To this means, two domestic pig types (German Landrace and hybrids), were compared to European wild boar. Along with clinical and pathological assessments and routine virological and serological methods, kinetics of immune-cellular parameters were evaluated. Findings All animals were susceptible to infection and despite clinical differences, virus could be detected in all infected animals to similar amounts. All but one animal developed an acute disease course, two landrace animals recovered after a transient infection. One wild boar got chronically infected. Changes in the percentages of lymphocyte subsets in peripheral blood did not show a clear correlation with the clinical outcome. High and early titers of neutralizing antibodies were especially detected in wild boar and German Landrace pigs. Conclusions While differences among breeds did not have the expected impact on course and outcome of CSFV infection, preload with facultative pathogens and even small differences in age seemed to be more relevant. Future studies will target the characterization of responses observed during different disease courses including cytokine reactions and further analyses of lymphocyte subsets.

Schmallenberg virus (SBV), a novel orthobunyavirus, was discovered in Germany in 2011. In adult ruminants SBV causes mild transient disease, but foetal infection can lead to severe malformations. Owing to its recent discovery, the knowledge about the pathogenesis is limited. In this study, two heifers seroconverted after a previous SBV infection and five SBV antibody-negative calves were subcutaneously inoculated, another two animals received SBV orally and three were kept as controls. In naive cattle infected subcutaneously viral RNA was detected in serum and blood samples for several days. Seropositive or orally inoculated animals as well as the uninfected controls remained negative throughout the study. Seroconversion was observed only after subcutaneous exposure of the naive animals to SBV. In lymphocytes from peripheral blood SBV genome was not detected, but the lymphocyte homeostasis in blood was influenced.

Classical swine fever virus (CSFV) C-strain “Riems” escape variants generated under selective antibody pressure with monoclonal antibodies and a peptide-specific antiserum in cell culture were investigated. Candidates with up to three amino acid exchanges in the immunodominant and highly conserved linear TAV-epitope of the E2-glycoprotein, and additional mutations in the envelope proteins ERNS and E1, were characterized both in vitro and in vivo. It was further demonstrated, that intramuscular immunization of weaner pigs with variants selected after a series of passages elicited full protection against lethal CSFV challenge infection. These novel CSFV C-strain variants with exchanges in the TAV-epitope present potential marker vaccine candidates. The DIVA (differentiating infected from vaccinated animals) principle was tested for those variants using commercially available E2 antibody detection ELISA. Moreover, direct virus differentiation is possible using a real-time RT-PCR system specific for the new C-strain virus escape variants or using differential immunofluorescence staining.

Highlights ► HaPyV VP1 protein based VLPs with GP33 CTL epitope were capable to induce CTL response in mice. ► VP1-GP33 VLPs were effectively processed in antigen presenting cells in vitro and in vivo. ► VP1-GP33 VLPs induced antigen-specific CD8+ T cell proliferation. ► VLPs-induced CTL responses had a protective and therapeutic capacity in mice in vivo. ► HaPyV VP1-derived VLP has potential for the induction of insert-specific CTL immune response., Hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) carrying model GP33 CTL epitope derived from Lymphocytic choriomeningitis virus (LCMV) were generated in yeast and examined for their capability to induce CTL response in mice. Chimeric VP1-GP33 VLPs were effectively processed in antigen presenting cells in vitro and in vivo and induced antigen-specific CD8+ T cell proliferation. Mice immunized only once with VP1-GP33 VLPs without adjuvant developed an effective GP33-specific memory T cell response: 70% were fully and 30% partially protected from LCMV infection. Moreover, aggressive growth of tumors expressing GP33 was significantly delayed in these mice in vivo. Therefore, HaPyV VP1-derived VLP harboring CTL epitopes are attractive vaccine candidates for the induction of insert-specific CTL immune response.

A licensed, inactivated vaccine based on a low pathogenic avian influenza virus strain (H5N2) was evaluated in layer hens kept under field conditions during a 2-year period. Vaccine efficacy was investigated by specific antibodies and by challenge-contact experiments using highly pathogenic avian influenza viruses (HPAIV) H5N1. Basic immunization with two applications induced clinical protection. Virus excretion by vaccinated hens was significantly reduced compared to non-vaccinated controls; transmission to non-vaccinated and vaccinated contact birds was not fully interrupted. Vaccination efficacy is influenced by several factors including antigenic relatedness between vaccine and field strains, but also by species, age and type of commercial uses of the host. Limitations and risks of HPAIV vaccination as silent spread of HPAIV and emergence of escape mutants must be considered a priori and appropriate corrective measures have to be installed.

Infectious laryngotracheitis is an important respiratory disease of chickens that is caused by an alphaherpesvirus [infectious laryngotracheitis virus (ILTV); Gallid herpesvirus 1]. As herpesvirus envelope glycoproteins are main targets of the humoral host immune response, they are of particular interest for development of vaccines, as well as of diagnostic tools. The conserved, N-glycosylated envelope protein gC has been identified as a major surface antigen of ILTV. To study the function of gC, we now isolated a gC-deleted ILTV recombinant as well as a gC rescuant after co-transfection of permissive chicken cells with virion DNA and transfer plasmids containing engineered subgenomic fragments. Like other alphaherpesvirus homologues, ILTV gC proved to be non-essential for replication. ILTV-DeltagC exhibited delayed penetration kinetics and slightly reduced plaque sizes in cultured chicken cells, whereas virus titres were not reduced significantly compared with wild-type or gC-rescued virus. In vivo studies revealed that ILTV-DeltagC is attenuated in chickens. However, infection with high doses of ILTV-DeltagC was still fatal for approximately 20 % of the animals, whereas wild-type or gC-rescued ILTV led to 50 % mortality. Interestingly, innate and specific immune responses against ILTV-DeltagC were not reduced but enhanced, and surviving chickens were protected completely against challenge infection. Furthermore, ILTV-DeltagC might serve as a basis for marker vaccines permitting differentiation between vaccinated and field-virus-infected animals, as gC-specific antibodies could be detected easily in sera of animals infected with wild-type ILTV.

Gene transfer into cells of mammalian, avian or piscine origin by baculoviruses carrying expression cassettes active in vertebrate cells (BacMam method) is an attractive alternative to chemical or physical transfection methods or to the use of vectors originating from viruses of vertebrates. For simultaneous high-level expression of two proteins from recombinant baculoviruses we constructed novel dual expression vectors containing human and murine cytomegalovirus immediate-early enhancer/promoter elements in combination with the baculoviral polyhedrin and p10 promoters for simultaneous expression in vertebrate and insect cells. Transduction of ruminant cells with BacMam viruses containing the green fluorescent protein open reading frame downstream from the respective enhancer/promoter elements revealed that a dual expression cassette combining the murine cytomegalovirus immediate-early 1 sequence with the immediate early enhancer/promoter of human cytomegalovirus yields high levels of protein from both transcription units. Protein expression directed by several cytomegalovirus/baculovirus hybrid promoters proceeded efficiently in insect cells infected with the respective recombinants. However, for expression in vertebrate cells the murine ie1 enhancer/promoter upstream the baculoviral p10 promoter was most efficient.

Item does not contain fulltext Adoptive transfer of tumor-specific T cells represents a promising approach for cancer immunotherapy. Here, we visualized the anti-tumor response of CD8 T cells from P14 TCR-transgenic mice specific for the model antigen GP33 by immunohistology. P14 T cells, adoptively transferred into tumor-bearing hosts, induced regression of established 3LL-A9(GP33) and MCA102(GP33) tumors that express GP33 as a tumor-associated model antigen. Strikingly, the visible effects of P14 T cell attack, such as the destruction of the tumor vasculature and accumulation of granulocytes, were predominantly detected inside the tumor mass. In regressing tumors, P14 T cells were found in the intact rim zone but not in central areas that were infiltrated with granulocytes and lacked CD31(+) endothelial cells. The rim of P14 T cell-treated tumors showed an increase in vascular density and decrease in hypoxia compared to untreated tumors. Hypoxic areas of tumors are known to exhibit decreased sensitivity to radiation therapy or chemotherapy. Thus, our data also imply that adoptive transfer of tumor-specific CD8 T cells might synergize with radiation therapy or chemotherapy in the elimination of solid tumors in vivo.

The characterisation of a monoclonal antibody (mab 45) reacting with phagocytic leucocytes isolated from blood and spleen of rainbow trout (Oncorhynchus mykiss, Walbaum) is described. The surface marker labelled by this mab is expressed at relative low levels on the membrane of large, nearly nongranulated trout leucocytes, and having the typical morphology of monocytes in flow cytometry (Kfoury et al., 1999, Fish Pathology, 34, 1-6). No reaction of mab 45 with granulocytes, lymphocytes or thrombocytes was detected. In spleen and head kidney, large, polymorphonuclear leucocytes were immunostained. The mab most strongly recognised an antigen of 48 kDa prepared from trout leucocytes of different organs, but not in trout plasma. In an in vitro phagocytosis assay trout monocytes were stained with mab 45 after phagocytosis of Aeromonas salmonicida labelled with the lipophilic fluorescent cell surface linker PKH26. However, previous binding of mab 45 on trout leucocytes did not inhibit the phagocytosis of A. salmonicida particles. Using mab 45, the dynamics of monocytes in blood, spleen and peritoneal cavity could be demonstrated after intraperitoneal injection of trout with inactivated A. salmonicida. The described mab serves as a useful tool to investigate the involvement of monocytes/macrophages in immune reactions of trout to a variety of pathogens.