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The invaluable health, economic and social impacts of vaccination are hard to exaggerate. The ability to stabilize vaccines is urgently required for their equitable distribution without the dependence on the 'cold-chain' logistics. Herein, for the first time we report biomimetic-mineralization of live-viral vaccines using metal-organic frameworks (MOFs) to enhance their storage stability from days to months. Applying ZIF-8 and aluminium fumarate (Alfum), the Newcastle Disease Virus (NDV) V4 strain and Influenza A WSN strain were encapsulated with remarkable retention of their viral titre. The ZIF-8@NDV, ZIF-8@WSN and Alfum@WSN composites were validated for live-virus recovery using a tissue culture infectious dose (TCID 50 ) assay. With the objective of long-term stabilization, we developed a novel, trehalose (T) and skim milk (SM) stabilized, freeze-dried MOF@Vaccine composite, ZIF-8@NDV+T/SM. The thermal stability of this composite was investigated and compared with the control NDV and non-encapsulated, freeze-dried NDV+T/SM composite at 4 °C, RT, and 37 °C over a period of 12 weeks. We demonstrate the fragility of the control NDV vaccine which lost all viability at RT and 37°C by 12 and 4 weeks, respectively. Comparing the freeze-dried counterparts, the MOF encapsulated ZIF-8@NDV+T/SM demonstrated significant enhancement in stability of the NDV+T/SM composite especially at RT and 37 °C upto 12 weeks. STATEMENT OF SIGNIFICANCE: Vaccination is undoubtedly one of the most effective medical interventions, saving millions of lives each year. However, the requirement of 'cold-chain' logistics is a major impediment to widespread immunization. Live viral vaccines (LVVs) are widely used vaccine types with proven efficacy and low cost. Nonetheless, their complex composition increases their susceptability to thermal stress. Several LVV thermostabilization approaches have been investigated, including their complex engineering and the facile addition of stabilizers. Still, the lack of a universal approach urgently requires finding a stabilization technique especially when additives alone may not be sufficient. Herein, we demonstrate MOF biomimetic-mineralization technology to encapsulate LVVs developing an optimised composite which significantly preserves vaccines without refrigeration for extended periods of time., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Chemotherapy-resistant cancer stem cells (CSCs) are a major obstacle to the effective treatment of many forms of cancer. To overcome CSC chemo-resistance, we developed a novel system by conjugating a CSC-targeting EpCAM aptamer with doxorubicin (Apt-DOX) to eliminate CSCs. Incubation of Apt-DOX with colorectal cancer cells resulted in high concentration and prolonged retention of DOX in the nuclei. Treatment of tumour-bearing xenograft mice with Apt-DOX resulted in at least 3-fold more inhibition of tumour growth and longer survival as well as a 30-fold lower frequency of CSC and a prolonged longer tumourigenic latency compared with those receiving the same dose of free DOX. Our data demonstrate that a CSC-targeting aptamer is able to transform a conventional chemotherapeutic agent into a CSC-killer to overcome drug resistance in solid tumours., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The ferret is an established animal model for a number of human respiratory viral infections, such as influenza virus and more recently, Ebola virus. However, a paucity of immunological reagents has hampered the study of cellular immune responses. Here we describe the development and characterisation of a novel monoclonal antibody (mAb) against the ferret CD4 antigen and the characterisation of ferret CD4 T lymphocytes. Recombinant production and purification of the ferret CD4 ectodomain soluble protein allowed hybridoma generation and the generation of a mAb (FeCD4) showing strong binding to ferret CD4 protein and lymphoid cells by flow cytometry. FeCD4 bound to its cognate antigen post-fixation with paraformaldehyde (PFA) which is routinely used to inactivate highly pathogenic viruses. We have also used FeCD4 in conjunction with other immune cell markers to characterise ferret T cells in both primary and secondary lymphoid organs. In summary, we have developed an important reagent for the study of cellular immunological responses in the ferret model of infectious disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The extracellular matrix (ECM) provides physical scaffolding for cellular constituents and initiates biochemical and biomechanical cues that are required for physiological activity of living tissues. The ECM enzyme ADAMTS5, a member of the ADAMTS (A Disintegrin-like and Metalloproteinase with Thrombospondin-1 motifs) protein family, cleaves large proteoglycans such as aggrecan, leading to the destruction of cartilage and osteoarthritis. However, its contribution to viral pathogenesis and immunity is currently undefined. Here, we use a combination of in vitro and in vivo models to show that ADAMTS5 enzymatic activity plays a key role in the development of influenza-specific immunity. Influenza virus infection of Adamts5-/- mice resulted in delayed virus clearance, compromised T cell migration and immunity and accumulation of versican, an ADAMTS5 proteoglycan substrate. Our research emphasises the importance of ADAMTS5 expression in the control of influenza virus infection and highlights the potential for development of ADAMTS5-based therapeutic strategies to reduce morbidity and mortality., Competing Interests: The authors have declared that no competing interests exist.

Hendra and Nipah viruses (family Paramyxoviridae, genus Henipavirus) are bat-borne viruses that cause fatal disease in humans and a range of other mammalian species. Gaining a deeper understanding of host pathways exploited by henipaviruses for infection may identify targets for new anti-viral therapies. Here we have performed genome-wide high-throughput agonist and antagonist screens at biosafety level 4 to identify host-encoded microRNAs (miRNAs) impacting henipavirus infection in human cells. Members of the miR-181 and miR-17~93 families strongly promoted Hendra virus infection. miR-181 also promoted Nipah virus infection, but did not affect infection by paramyxoviruses from other genera, indicating specificity in the virus-host interaction. Infection promotion was primarily mediated via the ability of miR-181 to significantly enhance henipavirus-induced membrane fusion. Cell signalling receptors of ephrins, namely EphA5 and EphA7, were identified as novel negative regulators of henipavirus fusion. The expression of these receptors, as well as EphB4, were suppressed by miR-181 overexpression, suggesting that simultaneous inhibition of several Ephs by the miRNA contributes to enhanced infection and fusion. Immune-responsive miR-181 levels was also up-regulated in the biofluids of ferrets and horses infected with Hendra virus, suggesting that the host innate immune response may promote henipavirus spread and exacerbate disease severity. This study is the first genome-wide screen of miRNAs influencing infection by a clinically significant mononegavirus and nominates select miRNAs as targets for future anti-viral therapy development., Competing Interests: The authors have declared that no competing interests exist.

Avian influenza viruses of H5 subtype can cause highly pathogenic disease in poultry. In March 2014, a new reassortant H5N6 subtype highly pathogenic avian influenza virus emerged in Lao People's Democratic Republic. We have assessed the pathogenicity, pathobiology and immunological responses associated with this virus in chickens. Infection caused moderate to advanced disease in 6 of 6 chickens within 48 h of mucosal inoculation. High virus titers were observed in blood and tissues (kidney, spleen, liver, duodenum, heart, brain and lung) taken at euthanasia. Viral antigen was detected in endothelium, neurons, myocardium, lymphoid tissues and other cell types. Pro-inflammatory cytokines were elevated compared to non-infected birds. Our study confirmed that this new H5N6 reassortant is highly pathogenic, causing disease in chickens similar to that of Asian H5N1 viruses, and demonstrated the ability of such clade 2.3.4-origin H5 viruses to reassort with non-N1 subtype viruses while maintaining a fit and infectious phenotype. Recent detection of influenza H5N6 poultry infections in Lao PDR, China and Viet Nam, as well as six fatal human infections in China, demonstrate that these emergent highly pathogenic H5N6 viruses may be widely established in several countries and represent an emerging threat to poultry and human populations., Competing Interests: The authors have declared that no competing interests exist.

Hendra and Nipah viruses (genus Henipavirus, family Paramyxoviridae) are highly pathogenic bat-borne viruses. The need for high biocontainment when studying henipaviruses has hindered the development of therapeutics and knowledge of the viral infection cycle. We have performed a genome-wide siRNA screen at biosafety level 4 that identified 585 human proteins required for henipavirus infection. The host protein with the largest impact was fibrillarin, a nucleolar methyltransferase that was also required by measles, mumps and respiratory syncytial viruses for infection. While not required for cell entry, henipavirus RNA and protein syntheses were greatly impaired in cells lacking fibrillarin, indicating a crucial role in the RNA replication phase of infection. During infection, the Hendra virus matrix protein co-localized with fibrillarin in cell nucleoli, and co-associated as a complex in pulldown studies, while its nuclear import was unaffected in fibrillarin-depleted cells. Mutagenesis studies showed that the methyltransferase activity of fibrillarin was required for henipavirus infection, suggesting that this enzyme could be targeted therapeutically to combat henipavirus infections.

While the ferret is a valuable animal model for a number of human viral infections, such as influenza, Hendra and Nipah, evaluating the cellular immune response following infection has been hampered by the lack of a number of species-specific immunological reagents. Interleukin 2 (IL-2) is one such key cytokine. Ferret recombinant IL-2 incorporating a C-terminal histidine tag was expressed and purified and the three-dimensional structure solved and refined at 1.89 Å by X-ray crystallography, which represents the highest resolution and first non-human IL-2 structure. While ferret IL-2 displays the classic cytokine fold of the four-helix bundle structure, conformational flexibility was observed at the second helix and its neighbouring region in the bundle, which may result in the disruption of the spatial arrangement of residues involved in receptor binding interactions, implicating subtle differences between ferret and human IL-2 when initiating biological functions. Ferret recombinant IL-2 stimulated the proliferation of ferret lymph node cells and induced the expression of mRNA for IFN-γ and Granzyme A., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Understanding the molecular basis of drug resistance and utilising this information to overcome chemoresistance remains a key challenge in oncology. Here we report that survivin, a key protein implicated in drug resistance, is overexpressed in cancer stem cell pool of doxorubicin-resistant breast cancer cells. Moreover, by utilising an active targeting system consisting of an RNA aptamer targeted against the epithelial cell adhesion molecule and a Dicer substrate survivin siRNA, we could deliver a high dose of the siRNA to cancer stem cells in xenograft tumours. Importantly, silencing of survivin with this aptamer-siRNA chimera in cancer stem cell population led to the reversal of chemoresistance, such that combined treatment with low dose of doxorubicin inhibited stemness, eliminated cancer stem cells via apoptosis, suppressed tumour growth, and prolonged survival in mice bearing chemoresistant tumours. This strategy for in vivo cancer stem cell targeting has wide application for future effective silencing of anti-death genes and in fact any dysregulated genes involved in chemoresistance and tumour relapse.

The identification of immune pathways that protect against pathogens may lead to novel molecular therapies for both livestock and human health. Interferon (IFN) is a major response pathway that stimulates multiple genes targeted towards reducing virus. Viperin is one such interferon stimulated gene (ISG) that helps protect mammals from virus and may be critical to protecting chickens in the same way. In chickens, ISGs are not generally well characterised and viperin, in concert with other ISGs, may be important in protecting against virus. Here we identify chicken viperin (ch-viperin) and show that ch-viperin is upregulated in response to viral signature molecules. We further show that viperin is upregulated in response to virus infection in vivo. This data will benefit investigators targeting the antiviral pathways in the chicken., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Emerging pathogenic viruses, such as avian influenza (AI), represent a serious threat to the poultry industry and human health. The development of novel therapeutics to protect against these viruses is critical and necessitates understanding the host immune mechanisms to find new pathways for protection against virus infection. Interferon (IFN) is a major antiviral arm of the immune system and is generally the first line of defence against virus. The multiple genes orchestrated by IFN upregulation are not well characterised in chickens due to a lack of reagents and research efforts. Here we have identified chicken ZAP (chZAP), an IFN stimulated gene (ISG), that has antiviral properties in human models, and show that chZAP is upregulated in response to PAMPs. Moreover, we show that chZAP is upregulated in vivo following particular viral infections. This data will benefit further studies that aim to understand antiviral response pathways in the chicken., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

Background: Bats are a major source of new and emerging viral diseases. Despite the fact that bats carry and shed highly pathogenic viruses including Ebola, Nipah and SARS, they rarely display clinical symptoms of infection. Host factors influencing viral replication are poorly understood in bats and are likely to include both pre- and post-transcriptional regulatory mechanisms. MicroRNAs are a major mechanism of post-transcriptional gene regulation, however very little is known about them in bats., Results: This study describes 399 microRNAs identified by deep sequencing of small RNA isolated from tissues of the Black flying fox, Pteropus alecto, a confirmed natural reservoir of the human pathogens Hendra virus and Australian bat lyssavirus. Of the microRNAs identified, more than 100 are unique amongst vertebrates, including a subset containing mutations in critical seed regions. Clusters of rapidly-evolving microRNAs were identified, as well as microRNAs predicted to target genes involved in antiviral immunity, the DNA damage response, apoptosis and autophagy. Closer inspection of the predicted targets for several highly supported novel miRNA candidates suggests putative roles in host-virus interaction., Conclusions: MicroRNAs are likely to play major roles in regulating virus-host interaction in bats, via dampening of inflammatory responses (limiting the effects of immunopathology), and directly limiting the extent of viral replication, either through restricting the availability of essential factors or by controlling apoptosis. Characterisation of the bat microRNA repertoire is an essential step towards understanding transcriptional regulation during viral infection, and will assist in the identification of mechanisms that enable bats to act as natural virus reservoirs. This in turn will facilitate the development of antiviral strategies for use in humans and other species.

Infection with H5N1 influenza virus is often fatal to poultry with death occurring in hours rather than days. However, whilst chickens may be acutely susceptible, ducks appear to be asymptomatic to H5N1. The mechanisms of disease pathogenesis are not well understood and the variation between different species requires investigation to help explain these species differences. Here we investigated the expression of several key proinflammatory cytokines of chickens and ducks following infection with 2 highly pathogenic H5N1 (A/Muscovy duck/Vietnam/453/2004 (Vt453) and A/Duck/Indramayu/BBVW/109/2006 (Ind109)) and a low-pathogenic H5N3 influenza virus (A/Duck/Victoria/1462/2008 (Vc1462)). H5N1 viruses caused fatal infections in chickens as well as high viral loads and increased production of proinflammatory molecules when compared to ducks. Cytokines, including Interleukin 6 (IL6) and the acute phase protein Serum Amyloid A (SAA), were rapidly induced at 24h post infection with H5N1. In contrast, low induction of these cytokines appeared in ducks and only at later times during the infection period. These observations support that hypercytokinemia may contribute to pathogenesis in chickens, whilst the lower cytokine response in ducks may be a factor in their apparent resistance to disease and decreased mortality., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Aim: Influenza virus remains a major threat, with outbreaks continuing to occur. Few treatment options are available and drug resistance can emerge rapidly. New drugs that can quickly be adapted to virus mutations are needed. Several highly effective siRNAs targeting influenza that inhibit virus replication are known; however, effective delivery of these siRNAs remains a challenge. The aim of this study was to demonstrate the safety and efficacy of ABA triblock copolymer-delivered siRNA to inhibit influenza virus replication in vivo., Materials & Methods: We report on the delivery of a siRNA targeting the influenza virus in chicken embryos using an ABA triblock copolymer prepared by reversible addition-fragmentation chain-transfer polymerization, containing a central cationic block and two outer hydrophilic polyethylene glycol blocks., Results: A significant reduction of virus titer was observed with the polymer/anti-influenza siRNA complexes, whereas the control with polymer/control siRNA complexes showed no effect., Conclusion: These data suggest that a reversible addition-fragmentation chain transfer-based siRNA delivery platform may be suitable for combating infectious diseases in vivo.

Zoonotic viruses that emerge from wildlife and domesticated animals pose a serious threat to human and animal health. In many instances, mouse models have improved our understanding of the human immune response to infection; however, when dealing with emerging zoonotic diseases, they may be of limited use. This is particularly the case when the model fails to reproduce the disease status that is seen in the natural reservoir, transmission species or human host. In this Review, we discuss how researchers are placing more emphasis on the study of the immune response to zoonotic infections in the natural reservoir hosts and spillover species. Such studies will not only lead to a greater understanding of how these infections induce variable disease and immune responses in distinct species but also offer important insights into the evolution of mammalian immune systems.

The prevalence of pathogenic viruses is a serious issue as they pose a constant threat to both the poultry industry and to human health. To prevent these viral infections an understanding of the host-virus response is critical, especially for the development of novel therapeutics. One approach in the control of viral infections would be to boost the immune response through administration of cytokines, such as interferons. However, the innate immune response in chickens is poorly characterised, particularly concerning the interferon pathway. This review will provide an overview of our current understanding of the interferon system of chickens, including their cognate receptors and known interferon-stimulated gene products., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The outcomes of viral infections are costly in terms of human and animal health and welfare worldwide. The observed increase in the virulence of some viruses and failure of many vaccines to stop these infections has lead to the apparent need to develop new anti-viral strategies. One approach to dealing with viral infection may be to employ the therapeutic administration of recombinant cytokines to act as 'immune boosters' to assist in augmenting the host response to virus. With this in mind, a greater understanding of the immune response, particularly cell mediated T-helper-1 (TH1) type responses, is imperative to the development of new anti-viral and vaccination strategies. Following the release of the chicken genome, a number of TH1-type cytokines have been identified, including chicken interleukin-12 (ChIL-12), ChIL-18 and interferon-γ ChIFN-γ), highlighting the nature of the TH1-type response in this non-mammalian vertebrate. To date a detailed analysis of the in vivo biological function of these cytokines has been somewhat hampered by access to large scale production techniques. This review describes the role of TH-1 cytokines in immune responses to viruses and explores their potential use in enhancing anti-viral treatment strategies in chickens. Furthermore, this review focuses on the example of ChIFN-γ treatment of Chicken Anemia Virus (CAV) infection. CAV causes amongst other things thymocyte depletion and thymus atrophy, as well as immunosuppression in chickens. However, due to vaccination, clinical disease appears less often, nevertheless, the subclinical form of the disease is often associated with secondary complicating infections due to an immunocompromised state. Since CAV-induced immunosuppression can cause a marked decrease in the immune response against other pathogens, understanding this aspect of the disease is critically important, as well as providing insights into developing new control approaches. With increasing emphasis on developing alternative control programs for poultry diseases, novel therapeutic strategies provide one approach. We show here that the in ovo administration of ChIFN-γ impacts the depletion of T-cell precursors during CAV infection. Therefore, it appears that ChIFN-γ may have the potential to be used as a novel therapeutic reagent to impact virus infection and alter immunosuppression caused by CAV and potentially other pathogens., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease.

Zoonotic viruses, such as highly pathogenic avian influenza (HPAI), present a significant threat to both the poultry industry and public health. The present method of controlling avian influenza (AI) relies on good farming practice with limited use of vaccination in some countries. However, new ways to control disease outbreaks might be possible with additional knowledge of the natural host response to virus. Moreover, manipulation of the innate immune system in mammals improves the outcomes following viral infection. A similar approach might be applied to the chicken, nevertheless, a greater knowledge of the chicken innate immune system is required. This review outlines important mammalian antiviral mechanisms that have been modulated to strengthen viral immunity and highlights the potential application of these strategies in the chicken, especially in regards, to AI., (Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.)

Newborn vertebrates may be susceptible to infection because the immature status of their immune system results in an inability to make an effective immune response. Consequently, newly hatched chicks appear to be more susceptible to infections than mature chickens. In particular, poultry susceptibility to virus infection may be related to poor expression of innate immune elements involved in antiviral responses. Therefore, in this study we assessed the relative development of the interferon (IFN) system: a protective system against virus infection. We investigated the age-related expression of the elements involved in the IFN response including IFN gene expression, their associated receptors and the pattern recognition receptors (PRR) involved in the regulation of IFNs. We observed that the IFN system is somewhat inadequately expressed in embryos and develops over time, just prior to and after hatching, and therefore chicks may be more susceptible to virus than mature birds because of an immature IFN network., (Crown Copyright © 2012. Published by Elsevier Ireland Ltd. All rights reserved.)

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.

Avian influenza virus is endemic in many regions around the world and remains a pandemic threat, a scenario tied closely to outbreaks of the virus in poultry. The innate immune system, in particular the nucleic acid-sensing toll-like receptors (TLRs) -3, -7, -8, and -9, play a major role in coordinating antiviral immune responses. In this study we have investigated the use of TLR ligands as antivirals against influenza A in chickens. The TLR7 ligand poly-C inhibited low-path influenza A growth in the chicken macrophage cell line HD-11 more effectively than poly(I:C), which acts via TLR3. The TLR7 ligand 7-allyl-8-oxoguanosine (loxoribine) inhibited influenza A replication in vitro and in ovo in a dose-dependent manner. Treatment of primary chicken splenocytes with loxoribine resulted in the induction of interferons-α, -β, and -λ, and interferon-stimulated genes PKR and Mx. These results demonstrate that nucleic acid-sensing TLR ligands show considerable potential as antivirals in chickens and could be incorporated into antiviral strategies.

The influence of in-feed lactoferrin (Lf) on bird production, intestinal microbiota, mucosal immune system and gut microarchitecture was assessed in male Cobb 500 broiler chickens. Birds were given one of four diets from day of hatch: Control (basal diet with no additives), ZnB (basal diet + 50 mg/kg zinc bacitracin), Lf 250 mg/kg (basal diet + 250 mg/kg Lf) and Lf 500 mg/kg (basal diet + 500 mg/kg Lf); n = 24 birds/treatment. An apparent metabolisable energy study was performed between d 25-32. Lf did not affect growth rate or feed conversion in the period 0-21 d of age, nor performance or energy metabolism during the 7 d metabolism experiment which commenced at 25 d of age.The profiles of caecal microbial communities were significantly different in birds given ZnB compared with birds given a diet with no additives, or supplemented with 250 mg/kg Lf. Birds given 250 mg/kg Lf also had a different microbial profile compared with birds given 500 mg/kg Lf. In comparison to control birds, Lf treated birds showed some differences in the T cell proportions in caecal tonsil and spleen. No differences in ileal villus height, crypt depth or goblet cell proportions were observed amongst dietary treatments. Whilst Lf had little effect on the measured parameters, the use of an integrated approach to study the influence of novel feed additives may facilitate a greater understanding of the relationships between nutrition, gut health and bird performance.

In mammals, Mda5 and RIG-I are members of the evolutionary conserved RIG-like helicase family that play critical roles in the outcome of RNA virus infections. Resolving influenza infection in mammals has been shown to require RIG-I; however, the apparent absence of a RIG-I homolog in chickens raises intriguing questions regarding how this species deals with influenza virus infection. Although chickens are able to resolve certain strains of influenza, they are highly susceptible to others, such as highly pathogenic avian influenza H5N1. Understanding RIG-like helicases in the chicken is of critical importance, especially for developing new therapeutics that may use these systems. With this in mind, we investigated the RIG-like helicase Mda5 in the chicken. We have identified a chicken Mda5 homolog (ChMda5) and assessed its functional activities that relate to antiviral responses. Like mammalian Mda5, ChMda5 expression is upregulated in response to dsRNA stimulation and following IFN activation of cells. Furthermore, RNA interference-mediated knockdown of ChMda5 showed that ChMda5 plays an important role in the IFN response of chicken cells to dsRNA. Intriguingly, although ChMda5 levels are highly upregulated during influenza infection, knockdown of ChMda5 expression does not appear to impact influenza proliferation. Collectively, although Mda5 is functionally active in the chicken, the absence of an apparent RIG-I-like function may contribute to the chicken's susceptibility to highly pathogenic influenza.

To better understand the immune response to highly pathogenic avian influenza virus, we compared expression of cytokines in chickens infected with avian influenza virus (A/Vietnam/1203/04) to that in uninfected chickens. Gene expression analyses revealed that influenza disseminated to multiple organs where immune responses could be identified. Among those cytokines influenced by influenza infection were the T helper type (Th)1-associated cytokines interleukin (IL)12 and interferon γ. In addition, a corresponding downregulation of the intracytoplasmic factor GATA3 was identified, whereas the Th2 cytokines IL4 and IL10 did not appear to be impacted by the infection. The inflammatory cytokine IL6 also appeared to be highly upregulated along with type 1 and type 3 interferon. Together, these data indicate that a strong inflammatory and Th1 response occurs after highly pathogenic avian influenza infection in the chicken that has implications for strategies that target the immune system for improving resistance to avian influenza.

Background: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis., Methodology/principal Findings: There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression., Conclusions/significance: The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies in the treatment of avian influenza infection.

Highly pathogenic avian influenza (HPAI) H5N1 virus is endemic in many regions around the world and remains a significant pandemic threat. To date H5N1 has claimed almost 300 human lives worldwide, with a mortality rate of 60% and has caused the death or culling of hundreds of millions of poultry since its initial outbreak in 1997. We have designed multi-functional RNA interference (RNAi)-based therapeutics targeting H5N1 that degrade viral mRNA via the RNAi pathway while at the same time augmenting the host antiviral response by inducing host type I interferon (IFN) production. Moreover, we have identified two factors critical for maximising the immunostimulatory properties of short interfering (si)RNAs in chicken cells (i) mode of synthesis and (ii) nucleoside sequence to augment the response to virus. The 5-bp nucleoside sequence 5'-UGUGU-3' is a key determinant in inducing high levels of expression of IFN-α, -β, -λ and interleukin 1-β in chicken cells. Positioning of this 5'-UGUGU-3' motif at the 5'-end of the sense strand of siRNAs, but not the 3'-end, resulted in a rapid and enhanced induction of type I IFN. An anti-H5N1 avian influenza siRNA directed against the PB1 gene (PB1-2257) tagged with 5'-UGUGU-3' induced type I IFN earlier and to a greater extent compared to a non-tagged PB1-2257. Tested against H5N1 in vitro, the tagged PB1-2257 was more effective than non-tagged PB1-2257. These data demonstrate the ability of an immunostimulatory motif to improve the performance of an RNAi-based antiviral, a finding that may influence the design of future RNAi-based anti-influenza therapeutics.

The recent European Union ban on the prophylactic use of in-feed antibiotics has escalated the search for alternatives for use within the poultry industry. When evaluating the efficacy of potential antibiotic alternatives on bird health and productivity, it is important to analyze the competence of the immune cells in the gut-associated lymphoid tissue (GALT), because it is routinely involved in the surveillance of colonizing microbes as well as in interacting with the ingested feed antigens. Therefore, we studied the effect of the prebiotics mannan-oligosaccharide (MOS) and fructo-oligosaccharide (FOS) on the phenotypic and functional competence of immune cells in cecal tonsil (CT), which is a major GALT. Day-old Cobb 500 male broilers were randomized to 4 groups. Control chickens were fed the basal diet only. Chickens in experimental groups received 0.05 g/kg zinc bacitracin or 5 g/kg of either FOS or MOS in addition to basal diet. At the end of 25 d, our comparison of the experimental groups with controls revealed that the addition of prebiotics to diet resulted in a significant reduction in the proportion of B cells and in mitogen responsiveness of lymphocytes in CT. Furthermore, FOS treatment significantly enhanced the IgM and IgG antibody titers in plasma. These findings emphasize the need for the analyses of the gut immune function following treatment with novel feed additives. The knowledge obtained from such analyses may aid in understanding the mechanisms underlying the immune competence of the birds, which needs consideration when selecting and optimizing new feed additives instead of antibiotics for poultry production.

Although Toll-like receptors (TLRs) have been well characterised in mammals, less work has been carried out in non-mammalian species, such as chickens. In this study the response of chicken cells to the TLR9 subfamily of ligands was characterised in vitro and in ovo. It was found that even though chickens appear to have only one functional receptor to represent the TLR9 subfamily, stimulation of chicken splenocytes with TLR7 and TLR9 ligands induced proinflammatory cytokine production and cell proliferation, similar to that observed when the homologous mammalian receptors are stimulated. Furthermore, we demonstrated that the in ovo administration of these TLR ligands elicits a response, such as cytokine production, that can be detected post-hatch. The current knowledge of the action of TLR ligands in mammals, in conjunction with their immunomodulating ability shown in this study, draws attention to their potential use as therapeutic agents for the poultry industry.

Zoonotic viruses, such as H5N1 Avian Influenza, pose major threats to both animals and humans, and with this in mind there is a need for the development of new anti-viral strategies. The cytokine interleukin-12 (IL-12) is known to play a pivotal regulatory role in the anti-viral response due to its role in the induction of the key anti-viral cytokine IFN-gamma. Therefore, strategies which provide a means for the production of therapeutic quantities of IL-12 may be of major benefit. Here we describe the development of biologically active Escherichia coli (E. coli) derived chicken IL-12 (ChIL-12). The single chain ChIL-12 gene was cloned into the pET32b expression vector, transformed into the BL-21 E. coli strain and expression induced with IPTG. Over expressed protein was solubilised with zwittergent detergent and isolated utilising Nickel ion affinity chromatography. Biological activity was determined as ChIL-12 stimulated proliferation of pre-treated T-cells in vitro. This study is the first example of a biologically active E. coli derived IL-12 from a non-mammalian vertebrate subsequently providing a means for testing the anti-viral therapeutic potential of ChIL-12 in an in vivo model.

The control of viral infections is of critical importance to livestock industries worldwide and is highlighted by costly infection outbreaks, such as that seen with foot and mouth disease virus. To ameliorate the impact of increasing problems with viral infections, new vaccine and anti-viral strategies are required and a greater understanding of the anti-viral response is essential. Furthermore, in pigs, evidence is still being gathered on the components of a defined anti-viral immune response. However, this has been greatly improved by the recent cloning and expression of critical cytokines involved in the anti-viral response. To assess the use of recombinant porcine interleukin-12 (rPoIL-12) as an immunotherapeutic and immunomodulator of swine, we have cloned and expressed rPoIL-12 as a single-chain fusion protein from Esherichia coli (E. coli). The fusion encodes the p40 and p35 subunits, linked by a glycine-serine linker and expressed as a C-terminal 6xHis tagged protein. rPoIL-12 stimulated the proliferation of human lymphoblasts and its activity on porcine cells was demonstrated by the ability of rPoIL-12 to increase the mRNA expression of porcine interleukin-18 receptor-alpha (poIL-18Ralpha) from porcine peripheral blood mononuclear cells (PoPMBCs). This data supports the inclusion of E. coli produced rPoIL-12 in immunomodulation strategies in the pig.

Increasing resistance to anthelmintic drugs indicates a vital need to develop alternative strategies to control helminth infections. Interleukin-3 (IL-3) is a multilineage hematopoietic growth regulator produced by activated T lymphocytes in response to infection. In helminth infections, eosinophils play an important role in the elimination of parasites through their recruitment of inflammatory cells and the release of granules. The ability of IL-3 to stimulate the development of eosinophils makes it a particularly important candidate for therapeutic use to protect against parasites. To enable the role of IL-3 in the development, growth, and differentiation of porcine eosinophils to be elucidated, recombinant IL-3 (rPoIL-3) was expressed and purified. As the amino acid sequence identities between porcine IL-3 and other reported species were quite low ( approximately 39% between human and pig), an assessment of the in vitro activity of rPoIL-3 was made. The culture of porcine bone marrow (BM) cells with rPoIL-3 stimulated the proliferation of SWC3a(hi) myeloid cells, conA rming that rPoIL-3 acted as a hematopoietic cell growth factor. Since rPoIL-3 stimulated the development of myeloid cells in culture, the in vivo potential to produce elevated eosinophil proportions was assessed. In vivo administration of rPoIL-3 induced a signiA cant increase in the number of eosinophils in blood. These results suggest that rPoIL-3 is a potent inducer of eosinophils in swine and supports the inclusion of rPoIL-3 in therapeutic strategies.

Background: Porcine dendritic cells (DC) are likely to be pivotal cells in the initiation of stimulatory and potential tolerogenic responses to xenoantigens, however, there are limited studies characterizing these antigen presenting cells., Methods: Porcine PBMC (CD172a(+)) were cultured with GM-CSF and IL-4 and phenotype and functional capabilities assessed. Lipopolysaccharide (LPS), IL-10, and IL-3 were added to the GM-CSF/IL-4 DC cultures to determine phenotypic and functional changes. Quantitative real-time polymerase chain reaction (PCR) for key cytokines was performed and the modified porcine DC were further assessed by primary mixed lymphocyte reaction to determine the effect of LPS, IL-10, and IL-3 on stimulatory capability., Results: Porcine PBMC (CD172(+)) cultured with GM-CSF and IL-4 produced cells with DC morphology, which were major histocompatability complex (MHC) class II(+), CD14(-/lo), and CD1a(lo). Addition of IL-10 or IL-3 to GM-CSF/IL-4 DC cultures produced cells with lower levels of MHC class II and higher levels of antigen uptake consistent with less mature DC. Quantitative real-time PCR of DC showed the addition of IL-10 induced an increase in IL-10 mRNA, no detectable IL-12, and reduced IL-6 mRNA. The addition of IL-3 to DC cultures decreased IL-12, IL-6 and tumor necrosis factor (TNF), with no change in IL-10 mRNA. GM-CSF/IL-4 DC induced strong human lymphocyte proliferation, compared with significantly reduced stimulatory capacity induced by IL-10 and IL-3 treated DC cultures., Conclusions: The profound effect on differential DC cytokine profile and reduced human anti-pig responses has important therapeutic implications in xenotransplantation. The mechanism of altered regulation warrants further investigation.

Interferons (IFN) provide a critical first line of defense against viral infection in vertebrates. Moreover, IFN-lambda, a recently identified group of mammalian IFN, has demonstrated antiviral potential in the treatment of mammalian viruses. With the growing concern over such diseases as avian influenza (AI), there is a pressing need for new antiviral strategies to manage problem viruses in poultry. Furthermore, the use of immune molecules, such as IFN-lambda, provides an attractive option for treating poultry by augmenting the host response to virus. With this in mind, we report here the first cloning, expression, and analysis of biologic activity of chicken IFN-lambda (ChIFN-lambda). We compared the similarity of ChIFN-lambda to those identified in other species and demonstrate that ChIFN-lambda has antiviral properties similar to those of human IFN-lambda (HuIFN-lambda). Our results demonstrate that in the chicken, as in human, the antiviral activity demonstrated by ChIFN-lambda supports its inclusion in therapeutic strategies directed against viral infections.

Toll-like receptors (TLRs) play key roles in the response to pathogens and in mammals the host response to virus critically relies on TLR3 to detect viral-derived dsRNA. However, in chickens there is a paucity of information about this pathway, and in view of the recent concerns with regard to highly pathogenic avian influenza, there is a clear need for understanding these antiviral pathways. Furthermore, TLR3 engagement is important to the outcome of viral infection because of its role in the induction of interferons (IFNs) and the diverse antiviral effects that these molecules induce. With this in mind, we have investigated the role of TLR3 and its impact on the production of IFNs. We show that in the chicken, poly(I:C), a dsRNA analogue, rapidly induces type 1 IFN similar to that seen in mammals. Furthermore, IFN can activate the upregulation of TLR3, which in some cell types induces them to become responsive to dsRNA. These data highlight the similar function that TLR3 plays in chickens and mammals. To determine the role of chicken TLR3 in response to poly(I:C), we used RNAi-mediated gene silencing to show that poly(I:C)-stimulated IFNbeta expression involves TLR3 signalling. The interrelationship between TLR3 and interferon as well as the observed increase in TLR3 and IFNbeta expression during H5N1 avian influenza infection indicates the importance of these molecules in viral infections in chickens.

Escherichia coli infection of the respiratory system in chickens occurs as a sequel to a variety of environmental stressors or microbial infections, culminating as chronic respiratory disease (CRD) syndrome or colibacillosis. These diseases cause significant production losses in poultry. With the growing concerns about the use of antibiotics in animal production, for diseases such as CRD, alternative natural agents, like cytokines, may be considered for enhancing health by stimulating the immune system. The current study was aimed at understanding the in vivo effects of recombinant chicken interferon-gamma (ChIFN-gamma) treatment on a variety of immunologic parameters during E. coli infection in chickens. Administration of ChIFN-gamma to chickens increased the percentage of phagocytes in lung and blood of E. coli-infected birds. At the phenotypic level, there was an increase in the percentage of cells expressing MHC II in the air sac, with a concomitant reduction in the proportion of these cells in blood. Furthermore, the blood plasma from ChIFN-gamma-treated infected birds showed an increased level of interleukin-6 (IL-6) activity. Cumulatively, these findings are indicative of in vivo enhancement of immune responses due to ChIFN-gamma. However, administration of ChIFN-gamma protein did not mitigate the development of air sac lesions following E. coli infection.

Viral infections in chickens pose a major health threat to the poultry industry. Infectious bronchitis virus (IBV) usually causes respiratory disease; however, the disease severity is influenced by the genotype of the chicken and the IBV strain involved. Nephropathogenic strains of IBV, such as the Australian T strain, can cause high mortalities due to kidney failure characterized by mononuclear cell infiltration and inflammation. In a previous study, a line of specific pathogen-free chickens, the S-line, was shown to be susceptible to high mortalities from IBV infection. The cause of these high mortalities is unknown but it is suspected that differential cytokine expression may play a role. With this in mind, we decided to study the role of the proinflammatory cytokine interleukin (IL)-6 during infection to determine its contribution to nephritis and influence on disease susceptibility. To investigate this, we infected the susceptible S-line and the more disease-resilient HWL line with the T strain of IBV and measured their cytokine response levels. In both lines of birds, IL-6 mRNA levels were elevated in the kidneys at 4 d postinfection. However, in S-line chickens, these levels were 20 times higher than those in the HWL chickens. In addition, S-line birds also showed three times higher serum IL-6 levels than HWL birds after IBV infection. These findings suggest that IL-6 may play a role in IBV-induced nephritis and may open an avenue to develop alternative strategies, such as the use of antiinflammatory cytokines, to overcome the nephropathogenic effects of IBV.

Viral diseases pose a significant threat to the poultry industry. However, there is currently a lack of antivirals and suitable vaccine adjuvants available to the poultry industry to combat this problem. The innate immune system is now recognised to be essential in the response to viral infection. However, in contrast to mammals, the innate immune response in chickens is relatively uncharacterised. The release of the full chicken genome sequence has accelerated the identification of genes involved in the immune response. The characterisation of these genes, including Toll-like receptors and cytokines has led to the identification of potential alternate antivirals and adjuvants., (Copyright 2007 S. Karger AG, Basel.)

Objectives: The isolation of porcine hematopoietic stem cells (HSC) would be an important step toward development of porcine-to-human chimerism for induction of tolerance in clinical xenotransplantation. CD34 is a common marker of HSC and has not been developed as a marker in pigs. In this study we have generated and characterized a monoclonal antibody (mAb) that identifies porcine CD34 on a subset of porcine bone marrow (BM) stem/progenitor cells., Methods: The porcine CD34 gene was cloned and a recombinant protein produced. An anti-porcine CD34 mAb was produced that could detect both the recombinant protein and a subset of porcine BM cells. The CD34(+) cells were phenotyped by lineage and HSC associated markers. Furthermore, the CD34(+) cells were analyzed by colony-forming unit (CFU) assay., Results: Two splice variants of the porcine CD34 gene were cloned and a recombinant protein produced for mAb production. The mAb developed can detect both the recombinant protein and the native CD34 protein on a range of pig tissues, including BM. This subset of BM cells was negative for hematopoietic lineage makers, including CD3, CD14, and CD21 and positive for other known porcine HSC markers, including CD90, CD172a, histocompatibility complex (MHC) class I, and MHC class II. Moreover, the CD34(+) BM cells were enriched for multilineage progenitor cells as determined by CFU assay., Conclusions: Similar to human and mouse CD34, pig CD34 detects a subset of BM progenitor cells. This mAb will now provide a means for isolating porcine CD34(+) cells to be further analyzed for HSC activity and to assess their potential to develop pig-to-human chimeras to induce xenograft tolerance.

Nucleic acid-induced gene silencing, such as RNA interference (RNAi), induces a multitude of responses in addition to the knockdown of a gene. This is best understood in the context of the antiviral immune response, from which the processes of RNAi are thought to be derived. Viral challenge of a vertebrate host leads to an intricate series of responses that orchestrate antiviral immunity. The success of this multifaceted system in overcoming viral encounters hinges on complex pathogen-host interactions. One aspect of these interactions, the nucleic acid-based immune response, is key to the successful resolution of a viral challenge. In particular, dsRNA, a nucleic acid associated with viral replication, is involved in numerous interactions contributing to induction, activation and regulation of antiviral mechanisms. Specifically, dsRNA is responsible for stimulating important protective responses, such as the activation of dicer-related antiviral pathways, induction of type 1 IFN, and stimulation of dsRNA-activated protein kinase and oligoadenylate synthetase. Furthermore, the modulation and shaping of this overall immune response is facilitated through nucleic acid interactions with pattern recognition receptors such as toll-like receptor 3. These diverse dsRNA-induced antiviral responses have implications for biotechnologies that use dsRNA to harness one arm of the host antiviral machinery for silencing a specific target gene. The interlinked nature of these response elements means that it may be difficult to completely isolate one element from the other arms of the antiviral response program of an organism. Thus, it is beneficial to understand all aspects of the immune response to dsRNA in order to manipulate these systems and minimize unwanted non-specific effects.

The worldwide trend towards a reduced reliance on in-feed antibiotics has increased the pressure to develop alternative strategies to manage infectious diseases in poultry. With this in mind, there is a great emphasis on vaccine use and the enhancement of existing vaccines to provide long-term protection. Currently existing adjuvants for poultry can have deleterious side-effects, such as inflammation, resulting in the down-grading of meat quality and a subsequent reduction in profits. Therefore, to enhance the use of vaccination, alternative adjuvants must be developed. The use of recombinant cytokines as adjuvants in poultry is attracting considerable attention, and their potential role as such has been addressed by several studies. The recent identification of a number of chicken cytokine genes has provided the possibility to study their effectiveness in enhancing the immune response during infection and vaccination. This review focuses on the recent studies involving the assessment of cytokines as vaccine adjuvants.

Cytokines, as immune activators, have been investigated in mammalian systems as natural adjuvants and therapeutics. In particular, interleukin-2 (IL-2) has been studied widely as a vaccine adjuvant and immuno-enhancer because of its role in activating T cell proliferation. We show here that the first nonmammalian IL-2 gene cloned, chicken IL-2 (ChIL-2), exhibits similar biologic activities to those of mammalian IL-2. To assess the activities of ChIL-2 in vivo, we injected birds with recombinant ChIL-2 (rChIL-2) protein. rChIL-2 treatment induced peripheral blood lymphocytes to express cell surface IL-2 receptors (IL-2R) within 48 h and resulted in an increase in the proportion of peripheral blood CD4+ and CD8+ T cells. Using bromodeoxyuridine (BrdU) incorporation as a measurement of cell proliferation, we showed the increase in T cell populations to be due to cell proliferation. The ability of ChIL-2 to cause both activation and proliferation of T cells in vivo indicates that it has the potential to be used as an immune activator.

Host immunity to mycobacterial infection is dependent on the activation of T lymphocytes and their recruitment with monocytes to form granulomas. These discrete foci of activated macrophages and lymphocytes provide a microenvironment for containing the infection. The cytokine, TNF, is essential for the formation and maintenance of granulomas, but the mechanisms by which TNF regulates these processes are unclear. We have compared the responses of TNF-deficient (TNF(-/-)) and wild-type C57BL/6 mice to infection with Mycobacterium smegmatis, a potent inducer of TNF, and virulent Mycobacterium tuberculosis to delineate the TNF-dependent and -independent components of the process. The initial clearance of M. smegmatis was TNF independent, but TNF was required for the early expression of mRNA encoding C-C and C-X-C chemokines and the initial recruitment of CD11b(+) macrophages and CD4(+) T cells to the liver during the second week of infection. Late chemokine expression and cell recruitment developed in TNF(-/-) mice associated with enhanced Th1-like T cell responses and mycobacterial clearance, but recruited leukocytes did not form tight granulomas. Infection of TNF(-/-) mice with M. tuberculosis also resulted in an initial delay in chemokine induction and cellular recruitment to the liver. Subsequently, increased mRNA expression was evident in TNF(-/-) mice, but the loosely associated lymphocytes and macrophages failed to form granulomas and prevent progressive infection. Therefore, TNF orchestrates early induction of chemokines and initial leukocyte recruitment, but has an additional role in the aggregation of leukocytes into functional granulomas capable of controlling virulent mycobacterial infection.

The use of antibiotic feed additives and chemical antimicrobials in food production animals is a double-edged sword. On one hand, it helps to prevent the outbreak of disease and promotes the growth of animals, but on the other hand, concerns are mounting over the emergence of antibiotic-resistant bacteria. As a consequence, some countries have already banned the use of in-feed antibiotics which has resulted in meat producers urgently seeking environmentally friendly alternative methods to control disease. Cytokines are proteins that control the type and extent of an immune response following infection or vaccination. They therefore represent excellent naturally occurring therapeutics. The use of cytokines in poultry has become more feasible with the discovery of a number of avian cytokine genes. Since the immune system of chickens is similar to that of mammals, they offer an attractive model system to study the effectiveness of cytokine therapy in the control of disease in livestock. This review will focus on the recent advances made in avian cytokines, with a particular focus on their assessment as therapeutic agents and vaccine adjuvants.

Mycobacterium tuberculosis infects humans through the lung, and immunity to this chronic infection is mediated primarily by CD4(+) T lymphocytes. Recently we have demonstrated that the recruitment of lymphocytes to the lung during primary aerosol M. tuberculosis infection in mice occurs predominantly through the interaction of alpha(4)beta(1) integrin on CD4(+) T cells and vascular cell adhesion molecule-1 on the pulmonary endothelium. To investigate the effect of route of immunization with Mycobacterium bovis BCG on the pattern of T-cell recruitment to the lung, we have analyzed the differences in expression of integrins on activated memory CD4(+) T cells infiltrating the lung following primary BCG immunization by aerosol, intravenous, and subcutaneous routes and after subsequent aerosol challenge with M. tuberculosis. There were marked differences in the patterns of recruitment of activated CD4(+) T cells to the lung following primary immunization by the three routes. Expansion of CD44(hi) CD62L(low) CD4(+) T cells in the lung occurred following aerosol and intravenous BCG immunizations, and the lymphocyte recruitment was proportional to the pulmonary bacterial load. The majority of infiltrating CD4(+) T cells expressed alpha(4)beta(1) integrin. On subsequent exposure to aerosol BCG rapid expansion of gamma interferon-secreting alpha(4)beta(1)(+) CD4(+) T cells occurred to the same extent in all immunized mice, regardless of the route of immunization. Similar expansion of alpha(4)beta(1)(+) CD4(+) memory T cells occurred following M. tuberculosis challenge. The three routes of BCG immunization resulted in the same level of protection against aerosol M. tuberculosis or BCG challenge in both the lungs and spleen. Therefore, recruitment of effector T lymphocytes and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior BCG immunization.

Interleukin (IL)-10 is an immunoregulatory cytokine that inhibits both Th1-like T cell responses and macrophage activation. Deficiency of IL-10 has been associated with increased Th1-like CD4+ T-cell responses and increased clearance of some intracellular pathogens, however, its role in mycobacterial infections is controversial. In order to examine the effects of mycobacterial virulence on the outcome of infection we compared infection with Mycobacterium avium and virulent Mycobacterium tuberculosis in C57Bl/6 IL-10-/- mice. M. avium infection in IL-10-/- mice resulted in sustained increases in interferon (IFN)-gamma-secreting T-cell responses and was associated with the increased clearance of M. avium from the liver and lung. By contrast, M. tuberculosis infection in IL-10-/- mice led to a transient increase in IFN-gamma T-cell responses at 4 weeks postinfection, with reduced bacterial burden in the lungs. This was not sustained so that by 8 weeks there was no difference to wild-type (WT) mice. In vitro infection of IL-10-/- macrophages with M. avium, but not M. tuberculosis, led to an increased IL-12 production. Therefore, endogenous IL-10 exerts a significant inhibition on specific IFN-gamma T-cell responses to M. avium infection, however, this effect is short lived during the M. tuberculosis infection, and fails to influence the long-term course of infection.

The resolution of pulmonary tuberculosis (TB) critically depends on the development of the Th1 type of immune responses, as exemplified by the exacerbation of TB in IL-12-deficient mice. Therefore, vaccination strategies optimizing IL-12 production by antigen-presenting cells (APC) in response to mycobacteria may have enhanced protective efficacy. Since dendritic cells (DC) are the critical APC for activation of CD4+ and CD8+ T cells, we examined whether stimulation of Mycobacterium bovis bacillus Calmette Guérin (BCG)-infected DC via CD40 increased their ability to generate Th1-oriented cellular immune responses. Incubation of DC with an agonistic anti-CD40 antibody activated CD40 signaling in DC, as shown by increased expression of major histocompatibility complex class II and costimulatory molecules, mRNA production for proinflammatory cytokines and interleukin 12 (IL-12) p40. This activation pattern was maintained when DC were stimulated with anti-CD40 antibody and infected with BCG. Importantly, CD40-stimulated BCG-infected DC displayed increased capacity to release bioactive IL-12 and to activate gamma interferon (IFN-gamma) producing T cells in vitro. Moreover, when C57BL/6 mice were immunized with these DC and challenged with aerosol Mycobacterium tuberculosis, increased levels of mRNA for IL-12 p40, IL-18, and IFN-gamma were present in the draining mediastinal lymph nodes. However, the mycobacterial burden in the lungs was not reduced compared to that in mice immunized with BCG-infected non-CD40-stimulated DC. Therefore, although the manipulation of DC via CD40 is effective for enhancing immune responses to mycobacteria in vivo, additional strategies are required to increase protection against virulent M. tuberculosis infection.