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1.  A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging 
PLoS ONE  2015;10(6):e0130375.
DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy.
PMCID: PMC4475043  PMID: 26091084
2.  Comparative analysis of enzymatically produced novel linear DNA constructs with plasmids for use as DNA vaccines 
Gene therapy  2014;21(7):645-652.
The use of DNA to deliver vaccine antigens offers many advantages, including ease of manufacture and cost. However, most DNA vaccines are plasmids and must be grown in bacterial culture, necessitating elements which are either unnecessary for effective gene delivery (e.g. bacterial origins of replication) or undesirable (e.g. antibiotic resistance genes). Removing these elements may improve the safety profile of DNA for the delivery of vaccines. Here we describe a novel, double-stranded, linear DNA construct produced by an enzymatic process that solely encodes an antigen expression cassette, comprising antigen, promoter, polyA tail and telomeric ends. We compared these constructs (called ‘Doggybones’ because of their shape) with conventional plasmid DNA. Using luciferase-expressing constructs, we demonstrated that expression levels were equivalent between Doggybones and plasmids both in vitro and in vivo. When mice were immunized with DNA constructs expressing the HIV envelope protein gp140, equivalent humoral and cellular responses were induced. Immunizations with either construct type expressing haemagluttinin were protective against H1N1 influenza challenge. This is the first example of an effective DNA vaccine which can be produced on a large scale by enzymatic processes.
PMCID: PMC4082409  PMID: 24830436
DNA Vaccine; Gene Delivery; Novel Construct
3.  Delayed Sequelae of Neonatal Respiratory Syncytial Virus Infection Are Dependent on Cells of the Innate Immune System 
Journal of Virology  2014;88(1):604-611.
Infection with respiratory syncytial virus (RSV) in neonatal mice leads to exacerbated disease if mice are reinfected with the same virus as adults. Both T cells and the host major histocompatibility complex genotype contribute to this phenomenon, but the part played by innate immunity has not been defined. Since macrophages and natural killer (NK) cells play key roles in regulating inflammation during RSV infection of adult mice, we studied the role of these cells in exacerbated inflammation following neonatal RSV sensitization/adult reinfection. Compared to mice undergoing primary infection as adults, neonatally sensitized mice showed enhanced airway fluid levels of interleukin-6 (IL-6), alpha interferon (IFN-α), CXCL1 (keratinocyte chemoattractant/KC), and tumor necrosis factor alpha (TNF-α) at 12 to 24 h after reinfection and IL-4, IL-5, IFN-γ, and CCL11 (eotaxin) at day 4 after reinfection. Weight loss during reinfection was accompanied by an initial influx of NK cells and granulocytes into the airways and lungs, followed by T cells. NK cell depletion during reinfection attenuated weight loss but did not alter T cell responses. Depletion of alveolar macrophages with inhaled clodronate liposomes reduced both NK and T cell numbers and attenuated weight loss. These findings indicate a hitherto unappreciated role for the innate immune response in governing the pathogenic recall responses to RSV infection.
PMCID: PMC3911760  PMID: 24173217
4.  Defining the Range of Pathogens Susceptible to Ifitm3 Restriction Using a Knockout Mouse Model 
PLoS ONE  2013;8(11):e80723.
The interferon-inducible transmembrane (IFITM) family of proteins has been shown to restrict a broad range of viruses in vitro and in vivo by halting progress through the late endosomal pathway. Further, single nucleotide polymorphisms (SNPs) in its sequence have been linked with risk of developing severe influenza virus infections in humans. The number of viruses restricted by this host protein has continued to grow since it was first demonstrated as playing an antiviral role; all of which enter cells via the endosomal pathway. We therefore sought to test the limits of antimicrobial restriction by Ifitm3 using a knockout mouse model. We showed that Ifitm3 does not impact on the restriction or pathogenesis of bacterial (Salmonella typhimurium, Citrobacter rodentium, Mycobacterium tuberculosis) or protozoan (Plasmodium berghei) pathogens, despite in vitro evidence. However, Ifitm3 is capable of restricting respiratory syncytial virus (RSV) in vivo either through directly restricting RSV cell infection, or by exerting a previously uncharacterised function controlling disease pathogenesis. This represents the first demonstration of a virus that enters directly through the plasma membrane, without the need for the endosomal pathway, being restricted by the IFITM family; therefore further defining the role of these antiviral proteins.
PMCID: PMC3836756  PMID: 24278312
5.  A “Prime-Pull” Vaccine Strategy Has a Modest Effect on Local and Systemic Antibody Responses to HIV gp140 in Mice 
PLoS ONE  2013;8(11):e80559.
One potential strategy for the prevention of HIV infection is to induce virus specific mucosal antibody that can act as an immune barrier to prevent transmission. The mucosal application of chemokines after immunisation, termed “prime-pull”, has been shown to recruit T cells to mucosal sites. We wished to determine whether this strategy could be used to increase B cells and antibody in the vaginal mucosa following immunisation with an HIV antigen. BALB/c mice were immunised intranasally with trimeric gp140 prior to vaginal application of the chemokine CCL28 or the synthetic TLR4 ligand MPLA, without antigen six days later. There was no increase in vaginal IgA, IgG or B cells following the application of CCL28, however vaginal application of MPLA led to a significant boost in antigen specific vaginal IgA. Follow up studies to investigate the effect of the timing of the “pull” stimulation demonstrated that when given 14 days after the initial immunisation MPLA significantly increased systemic antibody responses. We speculate that this may be due to residual inflammation prior to re-immunisation. Overall we conclude that in contrast to the previously observed effect on T cells, the use of “prime-pull” has only a modest effect on B cells and antibody.
PMCID: PMC3834027  PMID: 24260419
6.  Mucosal Application of gp140 Encoding DNA Polyplexes to Different Tissues Results in Altered Immunological Outcomes in Mice 
PLoS ONE  2013;8(6):e67412.
Increasing evidence suggests that mucosally targeted vaccines will enhance local humoral and cellular responses whilst still eliciting systemic immunity. We therefore investigated the capacity of nasal, sublingual or vaginal delivery of DNA-PEI polyplexes to prime immune responses prior to mucosal protein boost vaccination. Using a plasmid expressing the model antigen HIV CN54gp140 we show that each of these mucosal surfaces were permissive for DNA priming and production of antigen-specific antibody responses. The elicitation of systemic immune responses using nasally delivered polyplexed DNA followed by recombinant protein boost vaccination was equivalent to a systemic prime-boost regimen, but the mucosally applied modality had the advantage in that significant levels of antigen-specific IgA were detected in vaginal mucosal secretions. Moreover, mucosal vaccination elicited both local and systemic antigen-specific IgG+ and IgA+ antibody secreting cells. Finally, using an Influenza challenge model we found that a nasal or sublingual, but not vaginal, DNA prime/protein boost regimen protected against infectious challenge. These data demonstrate that mucosally applied plasmid DNA complexed to PEI followed by a mucosal protein boost generates sufficient antigen-specific humoral antibody production to protect from mucosal viral challenge.
PMCID: PMC3691144  PMID: 23826293
7.  Preexposure to CpG Protects against the Delayed Effects of Neonatal Respiratory Syncytial Virus Infection 
Journal of Virology  2012;86(19):10456-10461.
Severe respiratory viral infection in early life is associated with recurrent wheeze and asthma in later childhood. Neonatal immune responses tend to be skewed toward T helper 2 (Th2) responses, which may contribute to the development of a pathogenic recall response to respiratory infection. Since neonatal Th2 skewing can be modified by stimulation with Toll-like receptor (TLR) ligands, we investigated the effect of exposure to CpG oligodeoxynucleotides (TLR9 ligands) prior to neonatal respiratory syncytial virus (RSV) infection in mice. CpG preexposure was protective against enhanced disease during secondary adult RSV challenge, with a reduction in viral load and an increase in Th1 responses. A similar Th1 switch and reduction in disease were observed if CpG was administered in the interval between neonatal infection and challenge. In neonates, CpG pretreatment led to a transient increase in expression of major histocompatibility complex class II (MHCII) and CD80 on CD11c-positive cells and gamma interferon (IFN-γ) production by NK cells after RSV infection, suggesting that the protective effects may be mediated by antigen-presenting cells (APC) and NK cells. We conclude that the adverse effects of early-life respiratory viral infection on later lung health might be mitigated by conditions that promote TLR activation in the infant lung.
PMCID: PMC3457284  PMID: 22811525
8.  Glucopyranosyl Lipid Adjuvant (GLA), a Synthetic TLR4 Agonist, Promotes Potent Systemic and Mucosal Responses to Intranasal Immunization with HIVgp140 
PLoS ONE  2012;7(7):e41144.
Successful vaccine development against HIV will likely require the induction of strong, long-lasting humoral and cellular immune responses in both the systemic and mucosal compartments. Based on the known immunological linkage between the upper-respiratory and urogenital tracts, we explored the potential of nasal adjuvants to boost immunization for the induction of vaginal and systemic immune responses to gp140. Mice were immunized intranasally with HIV gp140 together with micellar and emulsion formulations of a synthetic TLR4 agonist, Glucopyranosyl Lipid Adjuvant (GLA) and responses were compared to R848, a TLR7/8 agonist, or chitosan, a non TLR adjuvant. GLA and chitosan but not R848 greatly enhanced serum immunoglobulin levels when compared to antigen alone. Both GLA and chitosan induced high IgG and IgA titers in nasal and vaginal lavage and feces. The high IgA and IgG titers in vaginal lavage were associated with high numbers of gp140-specific antibody secreting cells in the genital tract. Whilst both GLA and chitosan induced T cell responses to immunization, GLA induced a stronger Th17 response and chitosan induced a more Th2 skewed response. Our results show that GLA is a highly potent intranasal adjuvant greatly enhancing humoral and cellular immune responses, both systemically and mucosally.
PMCID: PMC3400629  PMID: 22829921
9.  Intravaginal immunization using the recombinant HIV-1 clade-C trimeric envelope glycoprotein CN54gp140 formulated within lyophilized solid dosage forms 
Vaccine  2011;29(27):4512-4520.
Vaccine-mediated prevention of primary HIV-1 infection at the heterosexual mucosal portal of entry may be facilitated by highly optimised formulations or drug delivery devices for intravaginal (i.vag) immunization. Previously we described hydroxyethylcellulose (HEC)-based rheologically structured gel vehicles (RSVs) for vaginal immunization of an HIV-1 vaccine candidate, a soluble recombinant trimeric HIV-1 clade-C envelope glycoprotein designated CN54gp140. Here we investigated the efficacy of lyophilized solid dosage formulations (LSDFs) for prolonging antigen stability and as i.vag delivery modalities. LSDFs were designed and developed that upon i.vag administration they would reconstitute with the imbibing of vaginal fluid to mucoadhesive, site-retentive semi-solids. Mice were immunized with lyophilized equivalents of (i) RSVs, (ii) modified versions of the RSVs more suited to lyophilization (sodium carboxymethyl cellulose (NaCMC)-based gels) and (iii) Carbopol® gel, all containing CN54gp140. NaCMC-based LSDFs provided significantly enhanced antigen stability compared to aqueous-based RSVs. Rheological analysis indicated the NaCMC-based LSDFs would offer enhanced vaginal retention in woman compared to more conventional vaginal gel formulations. All LSDFs were well tolerated in the mouse model. Following i.vag administration, all LSDFs boosted systemic CN54gp140-specific antibody responses in sub-cutaneously primed mice. Induction of CN54gp140-specific antibody responses in the female genital tract was evident. Of all the LSDFs the fastest releasing which was lyophilized Carbopol® gel elicited immune responses comparable to buffer instillation of antigen suggesting that rather than slower sustained release, initial high burst release from the LSDFs may suffice. The boosting of specific immune responses upon i.vag administration indicates that LSDFs are viable mucosal vaccine delivery modalities promoting antigen stability and facilitating intimate exposure of CN54gp140 to the mucosal-associated lymphoid tissue of the female genital tract.
PMCID: PMC3120965  PMID: 21514349
Intravaginal delivery; Mucosal vaccination; HIV-1; Lyophilized solid dosage formulations; DSC, differential scanning calorimetry; GNA, Galanthus nivalis; HEC, hydroxyethylcellulose; HPMC, hydroxypropylmethylcellulose; LSDFs, lyophilized solid dosage formulations; NaCMC, sodium carboxymethyl cellulose; PC, polycarbophil; PVP, polyvinylpyrollidone; RSVs, rheologically structured gel vehicles; SVF, simulated vaginal fluid
10.  CD25+ Natural Regulatory T Cells Are Critical in Limiting Innate and Adaptive Immunity and Resolving Disease following Respiratory Syncytial Virus Infection▿ †  
Journal of Virology  2010;84(17):8790-8798.
Regulatory CD4+ T cells have been shown to be important in limiting immune responses, but their role in respiratory viral infections has received little attention. Here we observed that following respiratory syncytial virus (RSV) infection, CD4+ Foxp3+ CD25+ natural regulatory T-cell numbers increased in the bronchoalveolar lavage fluid, lung, mediastinal lymph nodes, and spleen. The depletion of CD25+ natural regulatory T cells prior to RSV infection led to enhanced weight loss with delayed recovery that was surprisingly accompanied by increased numbers of activated natural killer cells in the lung and bronchoalveolar lavage fluid on day 8 postinfection. Increased numbers of neutrophils were also detected within the bronchoalveolar lavage fluid and correlated with elevated levels of myeloperoxidase as well as interleukin-6 (IL-6) and gamma interferon (IFN-γ). CD25+ natural regulatory T-cell depletion also led to enhanced numbers of proinflammatory T cells producing IFN-γ and tumor necrosis factor alpha (TNF-α) in the lung. Despite these increases in inflammatory responses and disease severity, the viral load was unaltered. This work highlights a critical role for natural regulatory T cells in regulating the adaptive and innate immune responses during the later stages of lung viral infections.
PMCID: PMC2919030  PMID: 20573822
11.  Respiratory Viral Infections in Infants: Causes, Clinical Symptoms, Virology, and Immunology 
Clinical Microbiology Reviews  2010;23(1):74-98.
Summary: In global terms, respiratory viral infection is a major cause of morbidity and mortality. Infancy, in particular, is a time of increased disease susceptibility and severity. Early-life viral infection causes acute illness and can be associated with the development of wheezing and asthma in later life. The most commonly detected viruses are respiratory syncytial virus (RSV), rhinovirus (RV), and influenza virus. In this review we explore the complete picture from epidemiology and virology to clinical impact and immunology. Three striking aspects emerge. The first is the degree of similarity: although the infecting viruses are all different, the clinical outcome, viral evasion strategies, immune response, and long-term sequelae share many common features. The second is the interplay between the infant immune system and viral infection: the immaturity of the infant immune system alters the outcome of viral infection, but at the same time, viral infection shapes the development of the infant immune system and its future responses. Finally, both the virus and the immune response contribute to damage to the lungs and subsequent disease, and therefore, any prevention or treatment needs to address both of these factors.
PMCID: PMC2806659  PMID: 20065326
12.  Delivery of Cytokines by Recombinant Virus in Early Life Alters the Immune Response to Adult Lung Infection▿  
Journal of Virology  2010;84(10):5294-5302.
Respiratory syncytial virus (RSV) is the main cause of bronchiolitis, the major cause of hospitalization of infants. An ideal RSV vaccine would be effective for neonates, but the immune responses of infants differ markedly from those of adults, often showing a bias toward T-helper 2 (Th2) responses and reduced gamma interferon (IFN-γ) production. We previously developed recombinant RSV vectors expressing IFN-γ and interleukin-4 (IL-4) that allow us to explore the role of these key Th1 and Th2 cytokines during infection. The aim of the current study was to explore whether an immunomodulation of infant responses could enhance protection. The expression of IFN-γ by a recombinant RSV vector (RSV/IFN-γ) attenuated primary viral replication in newborn mice without affecting the development of specific antibody or T-cell responses. Upon challenge, RSV/IFN-γ mice were protected from the exacerbated disease observed for mice primed with wild-type RSV; however, antiviral immunity was not enhanced. Conversely, the expression of IL-4 by recombinant RSV did not affect virus replication in neonates but greatly enhanced Th2 immune responses upon challenge without affecting weight loss. These studies demonstrate that it is possible to manipulate infant immune responses by using cytokine-expressing recombinant viruses and that neonatal deficiency in IFN-γ responses may lead to enhanced disease during secondary infection.
PMCID: PMC2863826  PMID: 20200251
13.  Interleukin 18 Coexpression during Respiratory Syncytial Virus Infection Results in Enhanced Disease Mediated by Natural Killer Cells▿  
Journal of Virology  2010;84(8):4073-4082.
Respiratory syncytial virus (RSV) causes bronchiolitis, the main cause of infantile hospitalization. Immunity against reinfection is poor, and there is great interest in boosting vaccine responses using live vectors expressing host cytokines. We therefore constructed a recombinant RSV expressing murine interleukin 18 (RSV/IL-18), a cytokine capable of inducing strong antiviral immune responses. In vitro RSV/IL-18 replicated at wild-type levels and produced soluble IL-18. In naïve BALB/c mice, RSV/IL-18 infection significantly increased both IL-18 mRNA and protein and attenuated the peak viral load 3-fold. Despite a reduced viral load, RSV/IL-18 infection caused a biphasic weight loss at days 2 and 6 postinfection that was not seen in wild-type infection. Day 2 disease was associated with enhanced pulmonary natural killer (NK) cell numbers and activity and was prevented by NK cell depletion during infection; day 6 disease was correlated with CD8 T-cell recruitment and was enhanced by NK cell depletion. IL-18 expression during priming also enhanced RSV-specific antibody responses and T-cell responses on secondary RSV infection. Therefore, while IL-18 boosted antiviral immunity and reduced the viral load, its coexpression worsened disease. This is the first recombinant RSV with this property, and these are the first studies to demonstrate that NK cells can induce pathology during pulmonary viral infections.
PMCID: PMC2849516  PMID: 20130064
14.  The Chemokine MIP1α/CCL3 Determines Pathology in Primary RSV Infection by Regulating the Balance of T Cell Populations in the Murine Lung 
PLoS ONE  2010;5(2):e9381.
CD8 T cells assist in the clearance of respiratory syncytial virus (RSV) infection from the lungs. However, disease after RSV infection is in part caused by excessive T cell activity, and a balance is therefore needed between beneficial and harmful cellular immune responses. The chemokine CCL3 (MIP1α) is produced following RSV infection and is broadly chemotactic for both T cells and natural killer (NK) cells. We therefore investigated its role in RSV disease.
Methodology/Principal Findings
CCL3 was produced biphasically, in both the early (day 1) and late (day 6–7) stages of infection. CCL3 depletion did not alter the recruitment of natural killer (NK) cells to the lungs during the early stage, but depletion did affect the later adaptive phase. While fewer T cells were recruited to the lungs of either CCL3 knockout or anti-CCL3 treated RSV infected mice, more RSV-specific pro-inflammatory T cells were recruited to the lung when CCL3 responses were impaired. This increase in RSV-specific pro-inflammatory T cells was accompanied by increased weight loss and illness after RSV infection.
CCL3 regulates the balance of T cell populations in the lung and can alter the outcome of RSV infection. Understanding the role of inflammatory mediators in the recruitment of pathogenic T cells to the lungs may lead to novel methods to control RSV disease.
PMCID: PMC2827540  PMID: 20195359
15.  Alveolar Macrophages Are a Major Determinant of Early Responses to Viral Lung Infection but Do Not Influence Subsequent Disease Development▿  
Journal of Virology  2008;82(9):4441-4448.
Macrophages are abundant in the lower respiratory tract. They play a central role in the innate response to infection but may also modulate excessive inflammation. Both macrophages and ciliated epithelial cells respond to infection by releasing soluble mediators, leading to the recruitment of innate and adaptive effector cells. To study the role of lung macrophages in acute respiratory viral infection, we depleted them by the inhalation of clodronate liposomes in an established mouse model of respiratory syncytial virus (RSV) disease. Infection caused an immediate local release of inflammatory cytokines and chemokines, peaking on day 1, which was virtually abolished by clodronate liposome treatment. Macrophage depletion inhibited the activation (days 1 to 2) and recruitment (day 4) of natural killer (NK) cells and enhanced peak viral load in the lung (day 4). However, macrophage depletion did not affect the recruitment of activated CD4 or CD8 T cells, weight loss, or virus-induced changes in lung function. Therefore, lung macrophages play a central role in the early responses to viral infection but have remarkably little effect on the adaptive response occurring at the time of peak disease severity.
PMCID: PMC2293049  PMID: 18287232
16.  The Role of T Cells in the Enhancement of Respiratory Syncytial Virus Infection Severity during Adult Reinfection of Neonatally Sensitized Mice▿  
Journal of Virology  2008;82(8):4115-4124.
Respiratory syncytial virus (RSV) is the major cause of infantile bronchiolitis and hospitalization. Severe RSV disease is associated with the development of wheezing in later life. In a mouse model of the delayed effects of RSV, the age at primary infection determines responses to reinfection in adulthood. During primary RSV infection, neonatal BALB/c mice developed only mild disease and recruited CD8 cells that were defective in gamma interferon production. Secondary reinfection of neonatally primed mice caused enhanced inflammation and profuse lung T-cell recruitment. CD4 cell depletion during secondary RSV challenge attenuated disease (measured by weight loss); depletion of CD8 cells also markedly attenuated disease severity but enhanced lung eosinophilia, and depletion of both CD4 and CD8 cells together completely abrogated weight loss. Depletion of CD8 (but not CD4) cells during primary neonatal infection was protective against weight loss during adult challenge. Therefore, T cells, in particular CD8 T cells, play a central role in the outcome of neonatal infection by enhancing disease during secondary challenge. These findings demonstrate a crucial role for T cells in the regulation of immune responses after neonatal infection.
PMCID: PMC2293007  PMID: 18272579
17.  Virally Delivered Cytokines Alter the Immune Response to Future Lung Infections▿  
Journal of Virology  2007;81(23):13105-13111.
Respiratory syncytial virus (RSV) is an important cause of infant morbidity and mortality worldwide and is increasingly recognized to have a role in the development and exacerbation of chronic lung diseases. There is no effective vaccine, and we reasoned that it might be possible to skew the immune system towards beneficial nonpathogenic responses by selectively priming protective T-cell subsets. We therefore tested recombinant RSV (rRSV) candidates expressing prototypic murine Th1 (gamma interferon [IFN-γ]) or Th2 (interleukin-4 [IL-4]) cytokines, with detailed monitoring of responses to subsequent infections with RSV or (as a control) influenza A virus. Although priming with either recombinant vector reduced viral load during RSV challenge, enhanced weight loss and enhanced pulmonary influx of RSV-specific CD8+ T cells were observed after challenge in mice primed with rRSV/IFN-γ. By contrast, rRSV/IL-4-primed mice were protected against weight loss during secondary challenge but showed airway eosinophilia. When rRSV/IL-4-primed mice were challenged with influenza virus, weight loss was attenuated but was again accompanied by marked airway eosinophilia. Thus, immunization directed toward enhancement of Th1 responses reduces viral load but is not necessarily protective against disease. Counter to expectation, Th2-biased responses were more beneficial but also influenced the pathological effects of heterologous viral challenge.
PMCID: PMC2169117  PMID: 17855518
18.  Role of CCL5 (RANTES) in Viral Lung Disease 
Journal of Virology  2006;80(16):8151-8157.
CCL5/RANTES is a key proinflammatory chemokine produced by virus-infected epithelial cells and present in respiratory secretions of asthmatics. To examine the role of CCL5 in viral lung disease, we measured its production during primary respiratory syncytial virus (RSV) infection and during secondary infection after sensitizing vaccination that induces Th2-mediated eosinophilia. A first peak of CCL5 mRNA and protein production was seen at 18 to 24 h of RSV infection, before significant lymphocyte recruitment occurred. Treatment in vivo with Met-RANTES (a competitive chemokine receptor blocker) throughout primary infection decreased CD4+ and CD8+ cell recruitment and increased viral replication. In RSV-infected, sensitized mice with eosinophilic disease, CCL5 production was further augmented; Met-RANTES treatment again reduced inflammatory cell recruitment and local cytokine production. A second wave of CCL5 production occurred on day 7, attributable to newly recruited T cells. Paradoxically, mice treated with Met-RANTES during primary infection demonstrated increased cellular infiltration during reinfection. We therefore show that RSV induces CCL5 production in the lung and this causes the recruitment of RSV-specific cells, including those making additional CCL5. If this action is blocked with Met-RANTES, inflammation decreases and viral clearance is delayed. However, the exact effects of chemokine modulation depend critically on time of administration, a factor that may potentially complicate the use of chemokine blockers in inflammatory diseases.
PMCID: PMC1563837  PMID: 16873271
19.  Differential Chemokine Expression following Respiratory Virus Infection Reflects Th1- or Th2-Biased Immunopathology 
Journal of Virology  2006;80(9):4521-4527.
Respiratory syncytial virus (RSV) is a major viral pathogen of infants that also reinfects adults. During RSV infection, inflammatory host cell recruitment to the lung plays a central role in determining disease outcome. Chemokines mediate cell recruitment to sites of inflammation and are influenced by, and influence, the production of cytokines. We therefore compared chemokine production in a mouse model of immunopathogenic RSV infection in which either Th1 or Th2 immunopathology is induced by prior sensitization to individual RSV proteins. Chemokine expression profiles were profoundly affected by the nature of the pulmonary immunopathology: “Th2” immunopathology in BALB/c mice was associated with increased and prolonged expression of CCL2 (MCP-1), CXCL10 (IP-10), and CCL11 (eotaxin) starting within 24 h of challenge. C57BL/6 mice with “Th2” pathology (enabled by a deficiency of CD8+ cells) also showed increased CCL2 production. No differences in chemokine receptor expression were detected. Chemokine blockers may therefore be of use for children with bronchiolitis.
PMCID: PMC1472012  PMID: 16611912
20.  Immune Responses and Disease Enhancement during Respiratory Syncytial Virus Infection 
Clinical Microbiology Reviews  2005;18(3):541-555.
Respiratory syncytial virus (RSV) is one of the commonest and most troublesome viruses of infancy. It causes most cases of bronchiolitis, which is associated with wheezing in later childhood. In primary infection, the peak of disease typically coincides with the development of specific T- and B-cell responses, which seem, in large part, to be responsible for disease. Animal models clearly show that a range of immune responses can enhance disease severity, particularly after vaccination with formalin-inactivated RSV. Prior immune sensitization leads to exuberant chemokine production, an excessive cellular influx, and an overabundance of cytokines during RSV challenge. Under different circumstances, specific mediators and T-cell subsets and antibody-antigen immune complex deposition are incriminated as major factors in disease. Animal models of immune enhancement permit a deep understanding of the role of specific immune responses in RSV disease, assist in vaccine design, and indicate which immunomodulatory therapy might be beneficial to children with bronchiolitis.
PMCID: PMC1195968  PMID: 16020689
21.  Role of CCL11 in Eosinophilic Lung Disease during Respiratory Syncytial Virus Infection 
Journal of Virology  2005;79(4):2050-2057.
Respiratory syncytial virus (RSV) is a major viral pathogen of infants and the elderly. Significant morbidity is caused by an overexuberant mixed lung cell infiltrate, which is thought to be driven by chemokines. One of the main chemotactic mediators responsible for the movement of eosinophils is CCL11 (eotaxin). Using a mouse model of eosinophilic bronchiolitis induced by RSV, we show here that treatment in vivo with a blocking antibody to CCL11 greatly reduces lung eosinophilia and disease severity. In addition, anti-CCL11 caused a striking inhibition of CD4-T-cell influx and shifted cytokine production away from interleukin-5 without reducing the resistance to viral replication. These results suggest that in addition to influencing eosinophil diapedesis and survival, anti-CCL11 has an action on T cells. These studies strengthen the case for anti-CCL11 treatment of Th2-driven diseases.
PMCID: PMC546549  PMID: 15681407
22.  Expression of tetanus toxin Fragment C in tobacco chloroplasts 
Nucleic Acids Research  2003;31(4):1174-1179.
Fragment C (TetC) is a non-toxic 47 kDa polypeptide fragment of tetanus toxin that can be used as a subunit vaccine against tetanus. Expression of TetC in Escherichia coli and yeast was dependent on the availability of synthetic genes that were required to improve translation efficiency and stabilize the mRNA. To explore the feasibility of producing TetC in tobacco leaves, we attempted expression of both the bacterial high-AT (72.3% AT) and the synthetic higher-GC genes (52.5% AT) in tobacco chloroplasts. We report here that the bacterial high-AT mRNA is stable in tobacco chloroplasts. Significant TetC accumulation was obtained from both genes, 25 and 10% of total soluble cellular protein, respectively, proving the versatility of plastids for expression of unmodified high-AT and high-GC genes. Mucosal immunization of mice with the plastid- produced TetC induced protective levels of TetC antibodies. Thus, expression of TetC in chloroplasts provides a potential route towards the development of a safe, plant-based tetanus vaccine for nasal and oral applications.
PMCID: PMC150239  PMID: 12582236
23.  Thymic stromal lymphopoietin (TSLP) acts as a potent mucosal adjuvant for HIV-1 gp140 vaccination in mice 
European Journal of Immunology  2011;42(2):353-363.
The development of a successful vaccine against HIV is likely to require the induction of strong and long-lasting humoral immune responses at the mucosal portal of virus entry. Hence, the design of a vaccine strategy able to induce mucosal antibodies and in particular specific IgA, may be crucial to providing immune protection. Nasal immunisation is known to induce specific IgG and IgA responses in the cervicovaginal mucosa; however, there is an urgent need for the development of safe, effective and accessible mucosal adjuvants for nasal application in humans. To reduce the potential for adverse events associated with some nasal adjuvants, we have assessed whether the B-cell-activating cytokines APRIL, BAFF and TSLP enhance humoral immune responses to HIV-1 gp140. Following intranasal immunisation, TSLP but not APRIL or BAFF induced strong humoral responses both in serum and mucosa. The adjuvant effect of TSLP on humoral responses was similar to that of cholera toxin (CT). The use of TSLP as an adjuvant skewed both the cellular and humoral immune responses towards Th2 cells. This is the first time that TSLP has been demonstrated to have a positive effect as a mucosal adjuvant, and specifically to promote mucosal and systemic responses to HIV gp140.
PMCID: PMC3378695  PMID: 22057556
Cytokine; HIV; Intranasal; Mucosal vaccine
24.  Metformin reduces airway glucose permeability and hyperglycaemia-induced Staphylococcus aureus load independently of effects on blood glucose 
Thorax  2013;68(9):835-845.
Diabetes is a risk factor for respiratory infection, and hyperglycaemia is associated with increased glucose in airway surface liquid and risk of Staphylococcus aureus infection.
To investigate whether elevation of basolateral/blood glucose concentration promotes airway Staphylococcus aureus growth and whether pretreatment with the antidiabetic drug metformin affects this relationship.
Human airway epithelial cells grown at air–liquid interface (±18 h pre-treatment, 30 μM–1 mM metformin) were inoculated with 5×105 colony-forming units (CFU)/cm2 S aureus 8325-4 or JE2 or Pseudomonas aeruginosa PA01 on the apical surface and incubated for 7 h. Wild-type C57BL/6 or db/db (leptin receptor-deficient) mice, 6–10 weeks old, were treated with intraperitoneal phosphate-buffered saline or 40 mg/kg metformin for 2 days before intranasal inoculation with 1×107 CFU S aureus. Mice were culled 24 h after infection and bronchoalveolar lavage fluid collected.
Apical S aureus growth increased with basolateral glucose concentration in an in vitro airway epithelia–bacteria co-culture model. S aureus reduced transepithelial electrical resistance (RT) and increased paracellular glucose flux. Metformin inhibited the glucose-induced growth of S aureus, increased RT and decreased glucose flux. Diabetic (db/db) mice infected with S aureus exhibited a higher bacterial load in their airways than control mice after 2 days and metformin treatment reversed this effect. Metformin did not decrease blood glucose but reduced paracellular flux across ex vivo murine tracheas.
Hyperglycaemia promotes respiratory S aureus infection, and metformin modifies glucose flux across the airway epithelium to limit hyperglycaemia-induced bacterial growth. Metformin might, therefore, be of additional benefit in the prevention and treatment of respiratory infection.
PMCID: PMC3756442  PMID: 23709760
Airway Epithelium; Bacterial Infection; COPD Exacerbations; Respiratory Infection

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