Correction: Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

[This corrects the article DOI: 10.1371/journal.ppat.1009330.].

Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans.

Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

Simultaneous Aerosol and Intramuscular Immunization with Influenza Vaccine Induces Powerful Protective Local T Cell and Systemic Antibody Immune Responses in Pigs.

A vaccine providing both powerful Ab and cross-reactive T cell immune responses against influenza viruses would be beneficial for both humans and pigs. In this study, we evaluated i.m., aerosol (Aer), and simultaneous systemic and respiratory immunization (SIM) by both routes in Babraham pigs, using the single cycle candidate influenza vaccine S-FLU. After prime and boost immunization, pigs were challenged with H1N1pdm09 virus. i.m.-immunized pigs generated a high titer of neutralizing Abs but poor T cell responses, whereas Aer induced powerful respiratory tract T cell responses but a low titer of Abs. SIM pigs combined high Ab titers and strong local T cell responses. SIM showed the most complete suppression of virus shedding and the greatest improvement in pathology. We conclude that SIM regimes for immunization against respiratory pathogens warrant further study.

Defining the Role of Cellular Immune Signatures in Diagnostic Evaluation of Suspected Tuberculosis.

BACKGROUND: Diagnosis of paucibacillary tuberculosis (TB) including extrapulmonary TB is a significant challenge, particularly in high-income, low-incidence settings. Measurement of Mycobacterium tuberculosis (Mtb)-specific cellular immune signatures by flow cytometry discriminates active TB from latent TB infection (LTBI) in case-control studies; however, their diagnostic accuracy and clinical utility in routine clinical practice is unknown. METHODS: Using a nested case-control study design within a prospective multicenter cohort of patients presenting with suspected TB in England, we assessed diagnostic accuracy of signatures in 134 patients who tested interferon-gamma release assay (IGRA)-positive and had final diagnoses of TB or non-TB diseases with coincident LTBI. Cellular signatures were measured using flow cytometry. RESULTS: All signatures performed less well than previously reported. Only signatures incorporating measurement of phenotypic markers on functional Mtb-specific CD4 T cells discriminated active TB from non-TB diseases with LTBI. The signatures measuring HLA-DR+IFNγ + CD4 T cells and CD45RA-CCR7-CD127- IFNγ -IL-2-TNFα + CD4 T cells performed best with 95% positive predictive value (95% confidence interval, 90-97) in the clinically challenging subpopulation of IGRA-positive but acid-fast bacillus (AFB) smear-negative TB suspects. CONCLUSIONS: Two cellular immune signatures could improve and accelerate diagnosis in the challenging group of patients who are IGRA-positive, AFB smear-negative, and have paucibacillary TB.

Establishment of a Pig Influenza Challenge Model for Evaluation of Monoclonal Antibody Delivery Platforms.

mAbs are a possible adjunct to vaccination and drugs in treatment of influenza virus infection. However, questions remain whether small animal models accurately predict efficacy in humans. We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing mAbs. We show that a strongly neutralizing mAb (2-12C) against the hemagglutinin head administered prophylactically at 15 mg/kg reduced viral load and lung pathology after pandemic H1N1 influenza challenge. A lower dose of 1 mg/kg of 2-12C or a DNA plasmid-encoded version of 2-12C reduced pathology and viral load in the lungs but not viral shedding in nasal swabs. We propose that the pig influenza model will be useful for testing candidate mAbs and emerging delivery platforms prior to human trials.

Innate activation of human primary epithelial cells broadens the host response to Mycobacterium tuberculosis in the airways.

Early events in the human airways determining whether exposure to Mycobacterium tuberculosis (Mtb) results in acquisition of infection are poorly understood. Epithelial cells are the dominant cell type in the lungs, but little is known about their role in tuberculosis. We hypothesised that human primary airway epithelial cells are part of the first line of defense against Mtb-infection and contribute to the protective host response in the human respiratory tract. We modelled these early airway-interactions with human primary bronchial epithelial cells (PBECs) and alveolar macrophages. By combining in vitro infection and transwell co-culture models with a global transcriptomic approach, we identified PBECs to be inert to direct Mtb-infection, yet to be potent responders within an Mtb-activated immune network, mediated by IL1ß and type I interferon (IFN). Activation of PBECs by Mtb-infected alveolar macrophages and monocytes increased expression of known and novel antimycobacterial peptides, defensins and S100-family members and epithelial-myeloid interactions further shaped the immunological environment during Mtb-infection by promoting neutrophil influx. This is the first in depth analysis of the primary epithelial response to infection and offers new insights into their emerging role in tuberculosis through complementing and amplifying responses to Mtb.

Stratification of Latent Mycobacterium tuberculosis Infection by Cellular Immune Profiling.

Background: Recently acquired and remotely acquired latent Mycobacterium tuberculosis infection (LTBI) are clinically indistinguishable, yet recent acquisition of infection is the greatest risk factor for progression to tuberculosis in immunocompetent individuals. We aimed to evaluate the ability of cellular immune signatures that differ between active tuberculosis and LTBI to distinguish recently from remotely acquired LTBI. Methods: Fifty-nine individuals were recruited: 20 had active tuberculosis, 19 had recently acquired LTBI, and 20 had remotely acquired LTBI. The proportion of mycobacteria-specific CD4+ T cells secreting tumor necrosis factor α (TNF-α) but not interferon γ or interleukin 2 which had a differentiated effector phenotype (TNF-α-only TEFF), and the level of CD27 expression on IFN-γ-producing CD4+ T cells, were detected by flow cytometry. Results: The TNF-α-only TEFF signature was significantly higher in the group with recently acquired LTBI, compared with the group with remotely acquired LTBI (P < .0001), and it discriminated between these groups with high sensitivity and specificity, with an area under the curve of 0.87. Two signatures incorporating CD27 expression did not distinguish between recently and remotely acquired LTBI. Interestingly, the TNF-α-only TEFF signature in participants with recently acquired LTBI was more similar to that in participants with tuberculosis than that in participants with remotely acquired LTBI, suggesting that recently acquired LTBI is immunologically more similar to tuberculosis than remotely acquired LTBI. Conclusions: These findings reveal marked biological heterogeneity underlying the clinically homogeneous phenotype of LTBI, providing a rationale for immunological risk stratification to improve targeting of LTBI treatment.

Langerhans cell histiocytosis is a neoplasm and consequently its recurrence is a relapse: In memory of Bob Arceci.

Langerhans cell histiocytosis (LCH) remains a poorly understood disorder with heterogeneous clinical presentations characterized by focal or disseminated lesions that contain excessive CD1a+ langerin+ cells with dendritic cell features known as "LCH cells." Two of the major questions investigated over the past century have been (i) the origin of LCH cells and (ii) whether LCH is primarily an immune dysregulatory disorder or a neoplasm. Current opinion is that LCH cells are likely to arise from hematopoietic precursor cells, although the stage of derailment and site of transformation remain unclear and may vary in patients with different extent of disease. Over the years, evidence has provided the view that LCH is a neoplasm. The demonstration of clonality of LCH cells, insufficient evidence alone for neoplasia, is now bolstered by finding driver somatic mutations in BRAF in up to 55% of patients with LCH, and activation of the RAS-RAF-MEK-ERK (where MEK and ERK are mitogen-activated protein kinase and extracellular signal-regulated kinase, respectively) pathway in nearly 100% of patients with LCH. Herein, we review the evidence that recurrent genetic abnormalities characterized by activating oncogenic mutations should satisfy prerequisites for LCH to be called a neoplasm. As a consequence, recurrent episodes of LCH should be considered relapsed disease rather than disease reactivation. Mapping the complete genetic landscape of this intriguing disease will provide additional support for the conclusion that LCH is a neoplasm and is likely to provide more potential opportunities for molecularly targeted therapies.

A novel murine cytomegalovirus vaccine vector protects against Mycobacterium tuberculosis.

Tuberculosis remains a global health problem so that a more effective vaccine than bacillus Calmette-Guérin is urgently needed. Cytomegaloviruses persist lifelong in vivo and induce powerful immune and increasing ("inflationary") responses, making them attractive vaccine vectors. We have used an m1-m16-deleted recombinant murine CMV (MCMV) expressing Mycobacterium tuberculosis Ag 85A to show that infection of mice with this recombinant significantly reduces the mycobacterial load after challenge with M. tuberculosis, whereas control empty virus has a lesser effect. Both viruses induce immune responses to H-2(d)-restricted epitopes of MCMV pp89 and M18 Ags characteristic of infection with other MCMVs. A low frequency of 85A-specific memory cells could be revealed by in vivo or in vitro boosting or after challenge with M. tuberculosis. Kinetic analysis of M. tuberculosis growth in the lungs of CMV-infected mice shows early inhibition of M. tuberculosis growth abolished by treatment with NK-depleting anti-asialo ganglio-N-tetraosylceramide Ab. Microarray analysis of the lungs of naive and CMV-infected mice shows increased IL-21 mRNA in infected mice, whereas in vitro NK assays indicate increased levels of NK activity. These data indicate that activation of NK cells by MCMV provides early nonspecific protection against M. tuberculosis, potentiated by a weak 85A-specific T cell response, and they reinforce the view that the innate immune system plays an important role in both natural and vaccine-induced protection against M. tuberculosis.

Accelerated in vivo proliferation of memory phenotype CD4+ T-cells in human HIV-1 infection irrespective of viral chemokine co-receptor tropism.

CD4(+) T-cell loss is the hallmark of HIV-1 infection. CD4 counts fall more rapidly in advanced disease when CCR5-tropic viral strains tend to be replaced by X4-tropic viruses. We hypothesized: (i) that the early dominance of CCR5-tropic viruses results from faster turnover rates of CCR5(+) cells, and (ii) that X4-tropic strains exert greater pathogenicity by preferentially increasing turnover rates within the CXCR4(+) compartment. To test these hypotheses we measured in vivo turnover rates of CD4(+) T-cell subpopulations sorted by chemokine receptor expression, using in vivo deuterium-glucose labeling. Deuterium enrichment was modeled to derive in vivo proliferation (p) and disappearance (d*) rates which were related to viral tropism data. 13 healthy controls and 13 treatment-naive HIV-1-infected subjects (CD4 143-569 cells/ul) participated. CCR5-expression defined a CD4(+) subpopulation of predominantly CD45R0(+) memory cells with accelerated in vivo proliferation (p = 2.50 vs 1.60%/d, CCR5(+) vs CCR5(-); healthy controls; P<0.01). Conversely, CXCR4 expression defined CD4(+) T-cells (predominantly CD45RA(+) naive cells) with low turnover rates. The dominant effect of HIV infection was accelerated turnover of CCR5(+)CD45R0(+)CD4(+) memory T-cells (p = 5.16 vs 2.50%/d, HIV vs controls; P<0.05), naïve cells being relatively unaffected. Similar patterns were observed whether the dominant circulating HIV-1 strain was R5-tropic (n = 9) or X4-tropic (n = 4). Although numbers were small, X4-tropic viruses did not appear to specifically drive turnover of CXCR4-expressing cells (p = 0.54 vs 0.72 vs 0.44%/d in control, R5-tropic, and X4-tropic groups respectively). Our data are most consistent with models in which CD4(+) T-cell loss is primarily driven by non-specific immune activation.

Will telomere erosion lead to a loss of T-cell memory?

Evidence is accumulating that elderly individuals are more susceptible to infection with organisms to which they were previously immune. This indicates that there might be a limit to the persistence of immune memory. This fact is particularly disturbing because the average life expectancy of humans has almost doubled in the past 200 years and is still increasing. We discuss mechanisms that might constrain the persistence of memory T cells and consider whether humans will suffer from memory T-cell exhaustion as life expectancy increases.

The Nikolas Symposia and histiocytosis.

Histiocytoses are a group of rare diseases that involve histiocytes (literally tissue cells (Greek), but in reality tissue-resident macrophages and dendritic cells), which are derived from bone-marrow stem cells. Histiocytoses pose problems similar to those of other rare diseases of childhood. Individual physicians see few cases, disease material is hard to collect and families suffer from lack of information and understanding. In this article, we describe how a series of 'think tank' meetings, the Nikolas Symposia, which have concentrated on Langerhans cell histiocytosis, have furthered our understanding of this enigmatic disease.

Pneumococcal conjugate and plain polysaccharide vaccines have divergent effects on antigen-specific B cells.

BACKGROUND: A 23-valent unconjugated pneumococcal polysaccharide vaccine (23vP), routinely administered at the age of 65, has limited effectiveness, and revaccination induces attenuated antibody responses. It is not known whether pneumococcal polysaccharide-protein conjugated vaccines (PCV), although highly effective in infants, offer any immunological advantages over 23vP in adults. METHODS: We immunized adults with schedules combining both PCV and 23vP and investigated B-cell responses to establish whether PCV7 (a 7-valent PCV) induced T-dependent responses in adults, to assess the role of memory B cells in 23vP-induced antibody hyporesponsiveness, and to identify the B-cell subtypes involved. RESULTS: A single dose of PCV7 induced significant increases in serotype-specific memory B-cell populations in peripheral blood indicating a T-dependent response. Conversely, immunization with 23vP resulted in a decrease in memory B-cell frequency. Furthermore, memory B-cell responses to subsequent immunization with PCV7, when given after 23vP, were attenuated. Notably, B1b cells, a subset important in protecting mice against pneumococci, were also depleted following immunization with 23vP in humans. CONCLUSIONS: This study indicates that PCV7 may have an immunological advantage over 23vP in adults and that 23vP-induced depletion of memory and B1b-cell subsets may provide a basis for antibody hyporesponsiveness and the limited effectiveness of 23vP. Clinical Trials Registration. ISRCTN: 78768849.

Spatial, temporal and molecular dynamics of swine influenza virus-specific CD8 tissue resident memory T cells.

For the first time we have defined naïve, central memory, effector memory and differentiated effector porcine CD8 T cells and analyzed their distribution in lymphoid and respiratory tissues after influenza infection or immunization, using peptide-MHC tetramers of three influenza nucleoprotein (NP) epitopes. The hierarchy of response to the three epitopes changes during the response in different tissues. Most NP-specific CD8 T cells in broncho-alveolar lavage (BAL) and lung are tissue resident memory cells (TRM) that express CD69 and downregulate CD45RA and CCR7. NP-specific cells isolated from BAL express genes characteristic of TRM, but gene expression differs at 7, 21 and 63 days post infection. In all tissues the frequency of NP-specific CD8 cells declines over 63 days almost to background levels but is best maintained in BAL. The kinetic of influenza specific memory CD8 T cell in this natural host species differs from that in small animal models.

CXCR6 is a marker for protective antigen-specific cells in the lungs after intranasal immunization against Mycobacterium tuberculosis.

Convincing correlates of protective immunity against tuberculosis have been elusive. In BALB/c mice, intranasal immunization with a replication-deficient recombinant adenovirus expressing Mycobacterium tuberculosis antigen 85A (adenovirus-85A) induces protective lower respiratory tract immunity against pulmonary challenge with Mycobacterium tuberculosis, while intradermal immunization with adenovirus-85A does not. Here we report that intranasal immunization with adenovirus-85A induces expression of the chemokine receptor CXCR6 on lung CD8 T lymphocytes, which is maintained for at least 3 months. CXCR6-positive antigen-specific T cell numbers are increased among bronchoalveolar lavage-recoverable cells. Similarly, intranasal immunization with recombinant antigen 85A with adjuvant induces CXCR6 expression on lung CD4 cells in BALB/c and C57BL/6 mice, while a synthetic ESAT6(1-20) peptide with adjuvant induces CXCR6 expression in C57BL/6 mice. Parenteral immunization fails to do so. Upregulation of CXCR6 is accompanied by a transient elevation of serum CXCL16 after intranasal immunization, and lung cells cultured ex vivo from mice immunized intranasally show increased production of CXCL16. Administration of CXCL16 and cognate antigen intranasally to mice previously immunized parenterally increases the number of antigen-specific T lymphocytes in the bronchoalveolar lavage-recoverable population, which mediates inhibition of the early growth of Mycobacterium tuberculosis after challenge. We conclude that expression of CXCR6 on lung T lymphocytes is a correlate of local protective immunity against Mycobacterium tuberculosis after intranasal immunization and that CXCR6 and CXCL16 play an important role in the localization of T cells within lung tissue and the bronchoalveolar lavage-recoverable compartment.

A randomized study comparing combined pneumococcal conjugate and polysaccharide vaccination schedules in adults.

BACKGROUND: The widely used 23-valent plain polysaccharide vaccine (23vP) has limited effectiveness, produces short-lived immune responses, and induces attenuated antibody production after subsequent challenge with pneumococcal vaccines. Our goal was to examine whether priming with the 7-valent pneumococcal conjugate vaccine (PCV7) could enhance the immunogenicity of 23vP for the PCV7 serotypes and to investigate whether 23vP induced hyporesponsiveness could be overcome using PCV7. METHODS: We conducted an open-label randomized study that compared 3 vaccine schedules, each of which consisted of 2 doses of PCV7 and 1 dose of 23vP (23vP-PCV7-PCV7, PCV7-23vP-PCV7, PCV7-PCV7-23vP) administered over a 1-year period in a cohort of 348 adults 50-70 years of age. All vaccines were administered intramuscularly and were given 6 months apart. Blood samples were obtained prior to and 1 month after each vaccination. RESULTS: 23vP administered after priming with 2 doses of PCV7 produced significantly higher antibody concentrations for 3 of the 7 PCV7 serotypes, compared with vaccination with a single dose of 23vP; however, the same immunogenicity could be achieved with a single dose of PCV7. Prior vaccination with 23vP attenuated the antibody response to subsequent PCV7, which was not restored by additional doses of PCV7. CONCLUSION: In adults, vaccination schedules combining PCV7 and 23vP do not provide improved immunogenicity over the use of a single dose of 23vP for most of the serotypes contained in PCV7.

Human cytomegalovirus-specific CD8(+) T-cell expansions contain long-lived cells that retain functional capacity in both young and elderly subjects.

The immune response to human cytomegalovirus (HCMV) infection is characterized by the accumulation of HCMV-specific CD8(+) T cells, particularly in the elderly; such expansions may impair immune responses to other pathogens. We investigated mechanisms underlying HCMV-specific expansions in 12 young and 21 old healthy subjects (although not all analyses were performed on all subjects). Phenotypically, HCMV-pentamer(+) CD8(+) T cells were characterized by marked Vß restriction, advanced differentiation (being predominantly CD27(-) CD28(-) ), and variable CD45RO/RA expression. Although more common and larger in older subjects, expansions had similar phenotypic characteristics in the young. In one old subject, repeated studies demonstrated stability in size and Vß distribution of pentamer(+) populations over 6 years. We tested whether HCMV-specific CD8(+) T-cell expansions arose from accelerated proliferation or extended lifespan by in vivo labelling with deuterated glucose and ex vivo Ki-67 expression. Uptake of deuterated glucose was lower in pentamer(+) cells than in pentamer(-) CD8(+) CD45RO(+) or CD8(+) CD45RA(+) cells in three old subjects, consistent with reduced proliferation and extended lifespan. Similarly Ki-67 labelling showed no evidence for increased proliferation in HCMV-specific CD8(+) expansions in older subjects, although pentamer(-) CD45RA(+) cells from young donors expressed very little Ki-67. We investigated Bcl-2 and CD95 as possible anti-apoptotic mediators, but neither was associated with pentamer-positivity. To investigate whether expansion represents a compensatory response to impaired functionality, we performed two tests of functionality, peptide-stimulated proliferation and CD107 expression; both were intact in pentamer(+) cells. Our data suggest that HCMV-specific CD8(+) expansions in older subjects accumulate by extended lifespan, rather than accelerated proliferation.