Severe asthma trajectories in adults: findings from the NORDSTAR cohort.

BACKGROUND: There is limited evidence on the pathways leading to severe asthma and we are presently unable to effectively predict the progression of the disease. We aimed to describe the longitudinal trajectories leading to severe asthma and to describe clinical events preceding disease progression in a nationwide population of patients with severe asthma. METHODS: We conducted an observational study based on Swedish data from the NORdic Dataset for aSThmA Research (NORDSTAR) research collaboration platform. We identified adult patients with severe asthma in 2018 according to the European Respiratory Society/American Thoracic Society definition and used latent class analysis to identify trajectories of asthma severity over a 10-year retrospective period from 2018. RESULTS: Among 169 128 asthma patients, we identified 4543 severe asthma patients. We identified four trajectories of severe asthma that were labelled as: trajectory 1 "consistently severe asthma" (n=389 (8.6%)), trajectory 2 "gradual onset severe asthma" (n=942 (20.7%)), trajectory 3 "intermittent severe asthma" (n=1685 (37.1%)) and trajectory 4 "sudden onset severe asthma" (n=1527 (33.6%)). "Consistently severe asthma" had a higher daily inhaled corticosteroid dose and more prevalent osteoporosis compared with the other trajectories. Patients with "gradual onset severe asthma" and "sudden onset severe asthma" developed type 2-related comorbidities concomitantly with development of severe asthma. In the latter group, this primarily occurred within 1-3 years preceding onset of severe asthma. CONCLUSIONS: Four distinct trajectories of severe asthma were identified illustrating different patterns of progression of asthma severity. This may eventually enable the development of better preventive management strategies in severe asthma.

Prevalence and management of severe asthma in the Nordic countries: findings from the NORDSTAR cohort.

Background: Real-life evidence on prevalence and management of severe asthma is limited. Nationwide population registries across the Nordic countries provide unique opportunities to describe prevalence and management patterns of severe asthma at population level. In nationwide register data from Sweden, Norway and Finland, we examined the prevalence of severe asthma and the proportion of severe asthma patients being managed in specialist care. Methods: This is a cross-sectional study based on the Nordic Dataset for Asthma Research (NORDSTAR) research collaboration platform. We identified patients with severe asthma in adults (aged ≥18 years) and in children (aged 6-17 years) in 2018 according to the European Respiratory Society/American Thoracic Society definition. Patients managed in specialist care were those with an asthma-related specialist outpatient contact (only available in Sweden and Finland). Results: Overall, we identified 598 242 patients with current asthma in Sweden, Norway and Finland in 2018. Among those, the prevalence of severe asthma was 3.5%, 5.4% and 5.2% in adults and 0.4%, 1.0%, and 0.3% in children in Sweden, Norway and Finland, respectively. In Sweden and Finland, 37% and 40% of adult patients with severe asthma and two or more exacerbations, respectively, were managed in specialist care; in children the numbers were 56% and 41%, respectively. Conclusion: In three Nordic countries, population-based nationwide data demonstrated similar prevalence of severe asthma. In children, severe asthma was a rare condition. Notably, a large proportion of patients with severe asthma were not managed by a respiratory specialist, suggesting the need for increased recognition of severe asthma in primary care.

Broadening of the T-cell repertoire to HIV-1 Gag p24 by vaccination of HLA-A2/DR transgenic mice with overlapping peptides in the CAF05 adjuvant.

Induction of broad T-cell immune responses is regarded as critical for vaccines against the human immunodeficiency virus type 1 (HIV-1) which exhibit high diversity and, therefore, focus has been on inducing cytotoxic CD8 T-cell responses against the more conserved parts of the virus, such as the Gag protein. Herein, we have used the p24 protein which contains a range of conserved T-cell epitopes. We demonstrate that a vaccine of HIV-1 subtype B consensus group-specific antigen (Gag) p24 protein with the CD8-inducing liposomal cationic adjuvant formulation (CAF) 05, induces both CD4 and CD8 T-cell responses in CB6F1 mice. The adjuvanted vaccine also induced functional antigen-specific cytotoxicity in vivo. Furthermore, we found that when fragmenting the Gag p24 protein into overlapping Gag p24 peptides, a broader T-cell epitope specificity was induced in the humanized human leukocyte antigen (HLA)-A2/DR-transgenic mouse model. Thus, combining overlapping Gag p24 peptides with CAF05 appears to be a promising and simple strategy for inducing broader T-cell responses to multiple conserved epitopes which will be relevant for both prophylactic and therapeutic HIV-1 vaccines.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M: leprae in Brazil, Ethiopia and Nepal

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Mycobacterium leprae virulence-associated peptides are indicators of exposure to M. leprae in Brazil, Ethiopia and Nepal.

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.

Development and preclinical safety evaluation of a new therapeutic HIV-1 vaccine based on 18 T-cell minimal epitope peptides applying a novel cationic adjuvant CAF01.

Therapeutic immunization of HIV-1-infected individuals with or without anti-retroviral therapy is a new promising disease prevention. To induce a new cytotoxic T(CD8) lymphocyte (CTL) immunity during chronic HIV-1 infection 15 infrequently targeted but conserved HLA-supertype binding CTL epitopes from Gag, Pol, Nef, Env, Vpu and Vif were identified. The 15 T(CD8) and three T(CD4) helper peptides were GMP synthesised and formulated with a new adjuvant CAF01 which is a synthetic two-component liposomic adjuvant comprising the quaternary ammonium dimethyl-dioctadecyl-ammonium (DDA) and the immune modulator trehalose 6,6'-dibehenate (TDB). Using IFN-γ ELISPOT assay, T-cell immune induction by the vaccine was found to both CD4 and CD8 T-cell restricted peptides in HLA-A2 transgenic mice. Comprehensive toxicity studies of the CAF01 adjuvant-alone and together with different vaccines showed that CAF01 when tested at human dose levels was safe and well tolerated with only local inflammation at the site of injection and no systemic reactions. No pharmacological safety issues were observed in Beagle dogs. The HIV-1 vaccine toxicity study in the Göttingen Minipig(®) showed no systemic toxicity from five repetitive i.m. injections, each with a 2-week interval, of either the 18 HIV-1 peptide antigen solution (AFO18) or the AFO18-CAF01, in which the 18 HIV-1 peptides were formulated with the CAF01 adjuvant. Distinct inflammatory responses were observed in the injected muscles of the AFO18-CAF01 vaccine treated animals as a result of the immune stimulating effect of the adjuvant on the vaccine. The results of the toxicity studies provide optimism for phase I clinical trials evaluating the therapeutic HIV-1 T-cell vaccination approach using multiple subdominant minimal epitope peptides applying the novel cationic adjuvant CAF01.

Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides.

Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.

Genome-based in silico identification of new Mycobacterium tuberculosis antigens activating polyfunctional CD8+ T cells in human tuberculosis.

Although CD8(+) T cells help control Mycobacterium tuberculosis infection, their M. tuberculosis Ag repertoire, in vivo frequency, and functionality in human tuberculosis (TB) remains largely undefined. We have performed genome-based bioinformatics searches to identify new M. tuberculosis epitopes presented by major HLA class I supertypes A2, A3, and B7 (covering 80% of the human population). A total of 432 M. tuberculosis peptides predicted to bind to HLA-A*0201, HLA-A*0301, and HLA-B*0702 (representing the above supertypes) were synthesized and HLA-binding affinities determined. Peptide-specific CD8(+) T cell proliferation assays (CFSE dilution) in 41 M. tuberculosis-responsive donors identified 70 new M. tuberculosis epitopes. Using HLA/peptide tetramers for the 18 most prominently recognized HLA-A*0201-binding M. tuberculosis peptides, recognition by cured TB patients' CD8(+) T cells was validated for all 18 epitopes. Intracellular cytokine staining for IFN-γ, IL-2, and TNF-α revealed mono-, dual-, as well as triple-positive CD8(+) T cells, indicating these M. tuberculosis peptide-specific CD8(+) T cells were (poly)functional. Moreover, these T cells were primed during natural infection, because they were absent from M. tuberculosis-noninfected individuals. Control CMV peptide/HLA-A*0201 tetramers stained CD8(+) T cells in M. tuberculosis-infected and noninfected individuals equally, whereas Ebola peptide/HLA-A*0201 tetramers were negative. In conclusion, the M. tuberculosis-epitope/Ag repertoire for human CD8(+) T cells is much broader than hitherto suspected, and the newly identified M. tuberculosis Ags are recognized by (poly)functional CD8(+) T cells during control of infection. These results impact on TB-vaccine design and biomarker identification.

HLA class I binding 9mer peptides from influenza A virus induce CD4 T cell responses.

BACKGROUND: Identification of human leukocyte antigen class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes from influenza virus is of importance for the development of new effective peptide-based vaccines. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, bioinformatics was used to predict 9mer peptides derived from available influenza A viral proteins with binding affinity for at least one of the 12 HLA-I supertypes. The predicted peptides were then selected in a way that ensured maximal coverage of the available influenza A strains. One hundred and thirty one peptides were synthesized and their binding affinities for the HLA-I supertypes were measured in a biochemical assay. Influenza-specific T cell responses towards the peptides were quantified using IFNgamma ELISPOT assays with peripheral blood mononuclear cells (PBMC) from adult healthy HLA-I typed donors as responder cells. Of the 131 peptides, 21 were found to induce T cell responses in 19 donors. In the ELISPOT assay, five peptides induced responses that could be totally blocked by the pan-specific anti-HLA-I antibody W6/32, whereas 15 peptides induced responses that could be completely blocked in the presence of the pan-specific anti-HLA class II (HLA-II) antibody IVA12. Blocking of HLA-II subtype reactivity revealed that 8 and 6 peptide responses were blocked by anti-HLA-DR and -DP antibodies, respectively. Peptide reactivity of PBMC depleted of CD4(+) or CD8(+) T cells prior to the ELISPOT culture revealed that effectors are either CD4(+) (the majority of reactivities) or CD8(+) T cells, never a mixture of these subsets. Three of the peptides, recognized by CD4(+) T cells showed binding to recombinant DRA1*0101/DRB1*0401 or DRA1*0101/DRB5*0101 molecules in a recently developed biochemical assay. CONCLUSIONS/SIGNIFICANCE: HLA-I binding 9mer influenza virus-derived peptides induce in many cases CD4(+) T cell responses restricted by HLA-II molecules.

Sequence conservation of subdominant HLA-A2-binding CTL epitopes in HIV-1 clinical isolates and CD8+ T-lymphocyte cross-recognition may explain the immune reaction in infected individuals.

Cytotoxic T-lymphocytes (CTL) are critical for immune control of infection with human immunodeficiency virus type-1 (HIV-1) and searches for relevant CTL epitopes for immune therapy are ongoing. Recently, we identified 28 HLA-A2-binding HIV-1 CTL epitopes (1). In this follow-up study we fully genome sequenced HIV-1 from 11 HLA-A2(+) patients to examine the sequence variation of these natural epitopes and compared them with the patient's CD8(+) T-cell recall response. Often the epitope was conserved but only a few patients showed a CD8(+) T-cell recall response. This infrequent targeting may be explained by immune subdominance. CD8(+) T-cell recall response to a natural epitope could be measured despite sequence differences in the patient's virus. T-cell cross-reaction between such variants could be demonstrated in HLA-A2 transgenic mice. Nine infrequently targeted but conserved or cross-reacting epitopes were identified in seven HIV-1 proteins. More immunogenic anchor amino acid optimized immunogens were designed that induced T-cell cross-reaction with these natural epitopes. It is concluded that most of the new CTL epitopes are conserved but subdominant during the infection. It is suggested that T-cell promiscuity may explain the observed CD8(+) T-cell reaction to epitope variants and it may be possible to use the selected immune optimized epitope peptides for therapeutic vaccination.

Inhibition of type I and type III interferons by a secreted glycoprotein from Yaba-like disease virus.

Type I (IFN-alpha/beta) and type III (IFN-lambdas) IFNs are important components of the host antiviral response. Although type III IFNs possess intrinsic antiviral activity similar to that of type I IFNs, they signal through a specific unique receptor complex, and their functional importance for antiviral resistance is largely uncharacterized. Here, we report the first virus defense mechanism that directly targets type III IFNs. Y136 from Yaba-like disease virus, a yatapoxvirus, is a secreted glycoprotein related to protein B18 from Vaccinia virus, a known type I IFN-binding protein and a member of the Ig superfamily. Surprisingly, whereas B18 inhibits only type I IFNs, Y136 inhibits both type I and type III IFNs. Y136 inhibits IFN-induced signaling and suppresses IFN-mediated biological activities including up-regulation of MHC class I antigen expression and induction of the antiviral state. These data demonstrate that poxviruses have developed unique strategies to counteract IFN-mediated antiviral protection and highlight the importance of type III IFNs in antiviral defense. These results suggest that type III IFNs may be an effective treatment for some poxviral infections.

CTL epitopes for influenza A including the H5N1 bird flu; genome-, pathogen-, and HLA-wide screening.

The purpose of the present study is to perform a global screening for new immunogenic HLA class I (HLA-I) restricted cytotoxic T cell (CTL) epitopes of potential utility as candidates of influenza A-virus diagnostics and vaccines. We used predictions of antigen processing and presentation, the latter encompassing 12 different HLA class I supertypes with >99% population coverage, and searched for conserved epitopes from available influenza A viral protein sequences. Peptides corresponding to 167 predicted peptide-HLA-I interactions were synthesized, tested for peptide-HLA-I interactions in a biochemical assay and for influenza-specific, HLA-I-restricted CTL responses in an IFN-gamma ELISPOT assay. Eighty-nine peptides could be confirmed as HLA-I binders, and 13 could be confirmed as CTL targets. The 13 epitopes, are highly conserved among human influenza A pathogens, and all of these epitopes are present in the emerging bird flu isolates. Our study demonstrates that present technology enables a fast global screening for T cell immune epitopes of potential diagnostics and vaccine interest. This technology includes immuno-bioinformatics predictors with the capacity to perform fast genome-, pathogen-, and HLA-wide searches for immune targets. To exploit this new potential, a coordinated international effort to analyze the precious source of information represented by rare patients, such as the current victims of bird flu, would be essential.

Characterization of near full-length genomes of HIV type 1 strains in Denmark: basis for a universal therapeutic vaccine.

We report here the near full-length sequence characterization of 17 Danish clinical HIV-1 strains isolated from HLA-A02 patients not in need of ART, with relatively low viral loads and normal CD4 cell counts. Sequencing was performed directly on DNA extracted from short-term cocultures of PBMCs. The near full-length genomes did not contain any major insertions, deletions, or rearrangements. Sixteen of the isolates were characterized as nonrecombinant subtype B and one isolate as nonrecombinant subtype C. Phylogenetic analysis did not reveal any founder effect among the sequences. Also, we investigated the presence of infrequently targeted subdominant HLA-A02-binding CTL epitopes. The epitopes were conserved in the Danish strains as well as globally in reference sequences of all subtypes. Thus, the selected epitopes were not subtype-specific or region-specific. This lends support for the concept of a universal immunotherapeutic vaccine construct based on these epitopes.

Optimization and immune recognition of multiple novel conserved HLA-A2, human immunodeficiency virus type 1-specific CTL epitopes.

MHC-I-restricted cytotoxic responses are considered a critical component of protective immunity against viruses, including human immunodeficiency virus type 1 (HIV-1). CTLs directed against accessory and early regulatory HIV-1 proteins might be particularly effective; however, CTL epitopes in these proteins are rarely found. Novel artificial neural networks (ANNs) were used to quantitatively predict HLA-A2-binding CTL epitope peptides from publicly available full-length HIV-1 protein sequences. Epitopes were selected based on their novelty, predicted HLA-A2-binding affinity and conservation among HIV-1 strains. HLA-A2 binding was validated experimentally and binders were tested for their ability to induce CTL and IFN-gamma responses. About 69 % were immunogenic in HLA-A2 transgenic mice and 61 % were recognized by CD8(+) T-cells from 17 HLA-A2 HIV-1-positive patients. Thus, 31 novel conserved CTL epitopes were identified in eight HIV-1 proteins, including the first HLA-A2 minimal epitopes ever reported in the accessory and regulatory proteins Vif, Vpu and Rev. Interestingly, intermediate-binding peptides of low or no immunogenicity (i.e. subdominant epitopes) were found to be antigenic and more conserved. Such epitope peptides were anchor-optimized to improve immunogenicity and further increase the number of potential vaccine epitopes. About 67 % of anchor-optimized vaccine epitopes induced immune responses against the corresponding non-immunogenic naturally occurring epitopes. This study demonstrates the potency of ANNs for identifying putative virus CTL epitopes, and the new HIV-1 CTL epitopes identified should have significant implications for HIV-1 vaccine development. As a novel vaccine approach, it is proposed to increase the coverage of HIV variants by including multiple anchor-optimized variants of the more conserved subdominant epitopes.