Landscape analysis of available European data sources amenable for machine learning and recommendations on usability for rare diseases screening.

BACKGROUND: Patient registries and databases are essential tools for advancing clinical research in the area of rare diseases, as well as for enhancing patient care and healthcare planning. The primary aim of this study is a landscape analysis of available European data sources amenable to machine learning (ML) and their usability for Rare Diseases screening, in terms of findable, accessible, interoperable, reusable(FAIR), legal, and business considerations. Second, recommendations will be proposed to provide a better understanding of the health data ecosystem. METHODS: In the period of March 2022 to December 2022, a cross-sectional study using a semi-structured questionnaire was conducted among potential respondents, identified as main contact person of a health-related databases. The design of the self-completed questionnaire survey instrument was based on information drawn from relevant scientific publications, quantitative and qualitative research, and scoping review on challenges in mapping European rare disease (RD) databases. To determine database characteristics associated with the adherence to the FAIR principles, legal and business aspects of database management Bayesian models were fitted. RESULTS: In total, 330 unique replies were processed and analyzed, reflecting the same number of distinct databases (no duplicates included). In terms of geographical scope, we observed 24.2% (n = 80) national, 10.0% (n = 33) regional, 8.8% (n = 29) European, and 5.5% (n = 18) international registries coordinated in Europe. Over 80.0% (n = 269) of the databases were still active, with approximately 60.0% (n = 191) established after the year 2000 and 71.0% last collected new data in 2022. Regarding their geographical scope, European registries were associated with the highest overall FAIR adherence, while registries with regional and "other" geographical scope were ranked at the bottom of the list with the lowest proportion. Responders' willingness to share data as a contribution to the goals of the Screen4Care project was evaluated at the end of the survey. This question was completed by 108 respondents; however, only 18 of them (16.7%) expressed a direct willingness to contribute to the project by sharing their databases. Among them, an equal split between pro-bono and paid services was observed. CONCLUSIONS: The most important results of our study demonstrate not enough sufficient FAIR principles adherence and low willingness of the EU health databases to share patient information, combined with some legislation incapacities, resulting in barriers to the secondary use of data.

Health-related quality of life among transthyretin amyloid cardiomyopathy patients.

AIMS: Transthyretin amyloid cardiomyopathy (ATTR CM) is a progressive and severe heart disease with physical and psychological implications. The Nordic PROACT study was conducted to investigate the health-related quality of life (HRQoL) in ATTR CM patients. METHODS AND RESULTS: The Nordic PROACT study was a cross-sectional non-interventional study conducted in 12 cardiology hospital clinics across Norway, Sweden, Finland and Denmark. Men and women aged ≥18 years diagnosed with symptomatic ATTR CM were included. The investigator provided information on medical history, biomarkers, current treatment, co-morbidities and disease severity according to the New York Heart Association (NYHA) class and the National Amyloidosis Centre (NAC) staging. Patients completed the HRQoL questionnaires in the form of the Kansas City Cardiomyopathy Questionnaire (KCCQ), the EQ-5D-5L index with Visual Analog Scale (VAS), and the Major Depression Inventory (MDI). A total of 169 patients (mean ± SD age 77.7 ± 6.2 years) were included. Ninety-two per cent were men. Seventy-six per cent had wildtype ATTR CM (ATTRwt CM) and 15% had a hereditary form of ATTR CM (ATTRv CM) while 9% were genetically unclassified. Most patients were in NYHA class II (54%) and NAC stage 1 (53%). Participation in randomized clinical trials (RCT) was noted in 58% of the patients. The 169 ATTR CM patients had a mean ± SD KCCQ score of 64.3 ± 23.1 for total symptom score, 64.8 ± 20.9 for overall summary score (OSS) and 65.1 ± 21.5 for clinical summary score. The EQ-5D-5L total utility score was 0.8 ± 0.2 and the EQ-5D-5L VAS score was 62.9 ± 20.6. The vast majority (89%) did not report any signs of depression. Patients with ATTRv CM had a higher KCCQ OSS as compared with ATTRwt CM, while EQ-5D-5L utility score, EQ-5D-5L VAS and MDI were similar. Non-RCT participants had a poorer HRQoL as compared with RCT participants as reflected in lower KCCQ OSS and EQ-5D-5L VAS scores and a higher MDI score. Patients with higher NYHA classes and NAC disease stages had a poorer HRQoL as demonstrated by lower KCCQ and EQ-5D-5L scores and higher MDI scores. Correlation between KCCQ, EQ-5D-5L and MDI and the covariate NYHA class remained significant (P < 0.05) after adjusting for multiple testing. CONCLUSIONS: KCCQ scores were lower than previously reported for patients with other heart diseases of non-ATTR CM origin. The HRQoL measures correlated well to NYHA class and NAC disease stage. The prevalence of depression appeared to be low.

Prevalence, characteristics, and mortality of patients with transthyretin amyloid cardiomyopathy in the Nordic countries.

AIMS: Transthyretin amyloid cardiomyopathy (ATTR-CM) is a progressive condition caused by deposition of transthyretin amyloid fibrils in the heart and is associated with poor quality of life and a shortened lifespan. This study aimed to describe the prevalence, clinical characteristics, and mortality of patients with ATTR-CM, using multiple national health registers in Denmark, Finland, Norway, and Sweden. METHODS AND RESULTS: Transthyretin amyloid cardiomyopathy patients were identified during 2008-2018 using a combination of diagnosis codes for amyloidosis and heart disease and were matched to patients with non-ATTR heart failure (HF). An identical study design was used in each country to facilitate comparison and aggregation of results. A total of 1930 ATTR-CM patients were identified from national health registers in the four countries. In 2018, prevalence of ATTR-CM per 100 000 inhabitants ranged from 1.4 in Denmark to 5.0 in Sweden; a steep increase over time was observed in Sweden and Norway. Median survival from diagnosis was 30 months for ATTR-CM patients and 67 months for matched HF patients. Survival was significantly lower for female than for male ATTR-CM patients (median survival: 22 and 36 months), while no significant difference was observed in the HF cohort. CONCLUSIONS: This study provides the first nationwide estimates of the prevalence, clinical characteristics, and mortality of patients with ATTR-CM, using identical study design across several countries. Findings corroborate previous case series showing high mortality in ATTR-CM, two-fold higher than for other HF patients and higher in women than men, highlighting the need for more precise and early diagnosis to reduce the disease burden.

Healthcare resource use of patients with transthyretin amyloid cardiomyopathy.

AIMS: Transthyretin amyloid cardiomyopathy (ATTR-CM) is the cardiac manifestation of transthyretin amyloidosis (ATTR). The aim of this study was to estimate healthcare resource use for ATTR-CM patients compared with heart failure (HF) patients, in Denmark, Finland, Norway, and Sweden. METHODS AND RESULTS: Data from nationwide healthcare registers in the four countries were used. ATTR-CM patients were defined as individuals diagnosed with amyloidosis and cardiomyopathy or HF between 2008 and 2018. Patients in the ATTR-CM cohort were matched to patients with HF but without ATTR-CM diagnosis. Resource use included number of visits to specialty outpatient and inpatient hospital care. A total of 1831 ATTR-CM and 1831 HF patients were included in the analysis. The mean number of hospital-based healthcare contacts increased in both the ATTR-CM and HF cohort during 3 years pre-diagnosis and was consistently higher for the ATTR-CM cohort compared with the HF cohort, with 6.1 [CI: 5.9-6.3] vs. 3.2 [CI: 3.1-3.3] outpatient visits and 1.03 [CI: 0.96-1.1] vs. 0.7 [CI: 0.7-0.8] hospitalizations. In the first year following diagnosis, patients with ATTR-CM continued to visit outpatient care (10.2 [CI: 10.1, 10.4] vs. 5.7 [CI: 5.6, 5.9]) and were admitted to hospital more frequently (3.3 [CI: 3.2, 3.4] vs. 2.5 [CI: 2.5, 2.6]) than HF patients. CONCLUSIONS: Transthyretin amyloid cardiomyopathy imposes a high burden on healthcare systems with twice as many outpatient specialist visits and 50% more hospitalizations in the year after diagnosis compared with HF patients without ATTR-CM. Studies to investigate if earlier diagnosis and treatment of ATTR-CM may lower resource use are warranted.

Nationwide prevalence and characteristics of transthyretin amyloid cardiomyopathy in Sweden.

OBJECTIVE: Transthyretin amyloid cardiomyopathy (ATTR-CM) is a rare, progressive and fatal condition caused by deposition of transthyretin amyloid fibrils in the heart. This study aims to identify all patients diagnosed with ATTR-CM in Sweden, estimate the prevalence of ATTR-CM, describe patient characteristics and mortality, assess the importance of early symptoms (red flags) for identification of ATTR-CM, and compare with patients with heart failure (HF). METHODS: This retrospective study combined multiple national health registers covering all specialist visits and prescriptions for the entire population of Sweden. Between January 2008 and December 2018, patients with ATTR-CM were identified retrospectively based on a combination of diagnosis codes and compared with matched, all-cause non-ATTR HF patients. RESULTS: Overall, a total of 994 patients diagnosed with ATTR-CM were identified, with an average age at diagnosis of 73 years, and 30% of whom were female. The prevalence of diagnosed ATTR-CM cases in 2018 was 5.0 per 100 000. The median survival from diagnosis was 37.6 months (CI 33.8 to 43.8), with a lower median survival in women (27.9 months, CI 23.3 to 33.8) compared with men (43.5 months, CI 37.6 to 49.6). Patients with ATTR-CM demonstrated reduced survival compared with patients with HF (p<0.001). Compared with patients with HF, clinical identification of carpal tunnel syndrome, spinal stenosis, and atrioventricular and left bundle branch block can facilitate earlier diagnosis of ATTR-CM. CONCLUSIONS: This study provides the first nationwide estimates of ATTR-CM prevalence and risk factors. The results reinforce the severity of the disease and the importance of earlier diagnosis, especially for female patients, in order to allow effective treatment and prevention of disease progression.

The capsular serotype of Streptococcus pneumoniae is more important than the genetic background for resistance to complement.

The polysaccharide capsule of Streptococcus pneumoniae inhibits phagocytic killing by innate immune mechanisms. Certain serotypes are associated with invasive disease while others with a nasopharyngeal carriage. The invasiveness of serotypes may partly be explained by ability to resist deposition of complement (C3) on the bacterial surface and consequent opsonophagocytic killing. In our previous studies, we observed that clinical isolates of serotypes 1 and 5, which are rarely detected in asymptomatic carriage, were resistant to complement deposition and opsonophagocytosis, whereas serotypes 6B and 23F, both common in carriage, were more sensitive to deposition of C3 and opsonophagocytic killing. However, presence of significant variation in C3 deposition between isolates of the same serotype indicated that factors other than the capsule also affect complement resistance. To distinguish the relative effect of the capsular serotype and other virulence factors on C3 deposition, we compared capsule-switched mutants prepared in genetic backgrounds of pneumococcal strains TIGR4, 603, and 618. Clinical isolates which had the same multilocus sequence type but expressed different serotypes were also compared. We found that the serotype had a significant impact on complement resistance and that the more resistant the strain was to complement, the higher was the concentration of polysaccharide-specific antibodies required for opsonophagocytic killing. Comparison of strains expressing the same capsular polysaccharides in the different genetic backgrounds and various capsular mutants of the same strain suggests that while the genotype affects complement resistance, the serotype is the most important determinant. Differences between serotypes were more significant than the differences between strains.

Serotype-related variation in susceptibility to complement deposition and opsonophagocytosis among clinical isolates of Streptococcus pneumoniae.

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae; it affects complement resistance and shields the bacterium from phagocytes. Certain capsular serotypes appear to be better able to cause invasive disease than others. Serotypes 1 and 5 are common causes of invasive disease but are rarely isolated from healthy carriers, whereas serotypes 6B and 23F are more frequently isolated from carriage than invasive disease. We have recently shown that serotypes 6B and 19F differ in resistance to complement C3 deposition and opsonophagocytic killing. In this study we assessed the complement resistance and susceptibility to opsonophagocytosis of several other serotypes targeted by the pneumococcal conjugate vaccines. Clinical isolates of serotypes 1, 4, 5, 14, 18C, and 23F were tested along reference strains of corresponding capsular types. The concentration of anticapsular antibodies required for opsonophagocytic killing correlated inversely with C3 deposition on the serotype. Serotype 1 was the most resistant of the clinical isolates to C3 deposition and, along with serotypes 5 and 19F, required the highest concentration of capsule antibodies for opsonophagocytic killing, whereas serotype 23F was the most sensitive to opsonophagocytosis. Sensitivity to C3 deposition and opsonophagocytosis was associated with serotype-specific mortality of invasive pneumococcal disease, suggesting that the primary pathogens, such as serotypes 1 and 5, are more resistant to complement and require a higher concentration of capsule antibodies for opsonophagocytic killing than the opportunistic serotypes such as 6B and 23F, which are associated with a more severe disease outcome.

Interaction of pneumococcal histidine triad proteins with human complement.

The pneumococcal histidine triad (Pht) proteins PhtA, PhtB, PhtD, and PhtE form a group of conserved pneumococcal surface proteins. Humans produce antibodies to Pht proteins upon exposure to pneumococcus, and immunization of mice has provided protective immunity against sepsis and pneumonia and reduced nasopharyngeal colonization. Pht proteins are candidates for inclusion in multicomponent pneumococcal protein vaccines. Their biological function in pneumococcal infections is not clear, but a role in complement inhibition has been suggested. We measured complement deposition on wild-type and Pht mutant strains in four genetic backgrounds: Streptococcus pneumoniae D39 (serotype 2) and R36A (unencapsulated derivative of D39) and strains of serotypes 3, 4, and 19F. PspA and PspC single and double mutants were compared to the wild-type and Pht-deficient D39 strains. Factor H binding was measured to bacterial cells, lysates, and protein antigens. Deletion of all four Pht proteins (Pht(-)) resulted in increased C3 deposition on the serotype 4 strain but not on the other strains. Pht antigens did not bind factor H, and deletion of Pht proteins did not affect factor H binding by bacterial lysates. The Pht(-) mutant serotype 4 strain bound slightly less factor H than the wild-type strain when binding was measured by flow cytometry. Pht proteins may play a role in immune evasion, but the mechanism of function is unlikely to be mediated by factor H binding. The relative contribution of Pht proteins to the inhibition of complement deposition is likely to be affected by the presence of other pneumococcal proteins and to depend on the genetic background.

Serotype-specific hyporesponsiveness to pneumococcal conjugate vaccine in infants carrying pneumococcus at the time of vaccination.

OBJECTIVE: To determine whether pneumococcal carriage at the time of 11-valent pneumococcal conjugate vaccine (PCV-11) administration interferes with immune response in infants. STUDY DESIGN: A total of 1111 Filipino infants recruited into an immunogenicity and carriage study, nested in an efficacy trial, received PCV-11 or saline solution placebo at 6, 10, and 14 weeks of age. Antibody concentrations to the most frequently carried vaccine serotypes 6B, 19F, and 23F were measured by enzyme immunoassay from sera obtained at 18 weeks and 9 months of age. Serotype-specific antibody concentration was compared between groups of children among PCV-11 recipients stratified according to their carriage status at 6 weeks of age. RESULTS: Antibody concentrations to 6B, 19F, and 23F were significantly lower at 18 weeks and 9 months of age among children who were carriers of the specific serotype at 6 weeks of age than among non-carriers of the serotype. The hyporesponsiveness was specific to the carried serotype. The specific antibody concentrations induced by PCV-11 among carriers did not differ significantly from those in placebo recipients, whereas the differences were highly significant among noncarriers. CONCLUSIONS: Pneumococcal carriage, prevalent in Filipino infants, interferes with serotype-specific immune response to primary series of PCV and has potential implications for immunization programs.

Streptococcus pneumoniae capsular serotype 19F is more resistant to C3 deposition and less sensitive to opsonophagocytosis than serotype 6B.

The polysaccharide capsule is a major virulence mechanism of Streptococcus pneumoniae, shielding the bacterium from phagocytes. Capsule types may differ in their abilities to resist immune defense. Antibody-mediated complement activation and opsonophagocytosis are crucial in protection against pneumococcus. Conjugate vaccine trials suggest imperfect protection against 19F. We have previously shown that significantly more anti-19F than anti-6B antibody is needed for killing in the opsonophagocytic assay (OPA). In this study, we explored whether the amount of C3 deposited on serotype 6B and 19F pneumococcal strains reflects their sensitivity to opsonophagocytosis. We compared clinical 6B and 19F nasopharyngeal, middle ear, and blood isolates as well as reference OPA strains (n = 16) for their sensitivity to opsonophagocytosis and C3 deposition. Sixfold anticapsular antibody concentrations were required for 50% opsonophagocytic killing of 19F compared to that of 6B strains. Serotype 19F was more resistant to C3 deposition than 6B. Complement deposition and opsonophagocytosis were dependent on the concentration of anticapsular antibodies. Differences between pneumococcal serotypes in antibody-mediated protection may partly be explained by the abilities of the capsules to resist complement deposition. These findings support previous studies suggesting that higher antibody concentrations to the capsular polysaccharide are needed for protection against disease caused by serotype 19F than that caused by 6B.

Cost-effectiveness of routine measuring of serum drug concentrations and anti-drug antibodies in treatment of rheumatoid arthritis patients with TNF-α blockers.

Monitoring of anti-drug antibodies (ADAbs) or serum concentrations of biologicals in treatment of rheumatoid arthritis could provide an explanation for a loss of efficacy and help in the choice of subsequent medication. Current clinical practices do not generally include such monitoring of tumor necrosis factor (TNF)-α blockers on a routine basis. The main aims of this study were to estimate the probabilities of optimal and nonoptimal treatment decisions if infliximab or adalimumab drug trough level (DL) and ADAbs are tested or not in rheumatoid arthritis, and to model cost-effectiveness of performing such monitoring on a routine basis. Data on DLs and ADAbs concentrations were obtained in Finland from clinically requested monitoring analyses of 486 and 1,137 samples from patients on adalimumab and infliximab, respectively. DL was within the target range in 42% of samples from adalimumab- and 50.4% of infliximab-treated patients. ADAbs were detected in approximately 20% and 13.5% of samples from adalimumab- and infliximab-treated patients, respectively. ADAbs were found in 52.3% and 41.3% of those with low adalimumab or infliximab DLs, respectively. The monitoring data were incorporated into probabilities for making the optimal treatment decision. Economic impact of clinical decision-making was modeled in a short-term (3-6 months) scenario with 100 hypothetical patients. In the model, the combined measurement of DLs and ADAbs was cost-saving compared to the nontesting scenario when the monitoring results affected the treatment decision in at least 2-5 of 100 patients, a proportion which is easily exceeded in real-life clinical practice. This study indicates that routine monitoring of drug level and ADAbs is cost-beneficial in clinical practice, thereby improving the decision-making process in using TNF-α blockers.

Aging reduces the functionality of anti-pneumococcal antibodies and the killing of Streptococcus pneumoniae by neutrophil phagocytosis.

We evaluated the effect of aging on the functional activity of naturally acquired anti-pneumococcal antibodies, the function of neutrophils in phagocytic killing of opsonized pneumococci, and the complement activity. Opsonic activities of antibodies to all tested pneumococcal serotypes were significantly lower and phagocytic killing of pneumococci by neutrophils was significantly impaired among the elderly, whereas the complement activity was slightly higher in the elderly than in the young adults. The reduced functional activity of serotype-specific antibodies and the compromised function of neutrophils in the opsonophagocytosis of pneumococci are likely to contribute to the increased susceptibility of the elderly to pneumococcal diseases.

Effects of ageing and gender on naturally acquired antibodies to pneumococcal capsular polysaccharides and virulence-associated proteins.

Elderly individuals are susceptible to pneumococcal infections. Although factors contributing to the increased susceptibility of the elderly to bacterial infections may be several, compromised immune function, a consequence of normal human ageing, is widely accepted to play a role. We evaluated the effect of ageing on the concentrations of naturally acquired antibodies to pneumococcal capsular polysaccharides (PPS) and protein antigens. The concentrations of immunoglobulin G (IgG) and IgM antibodies to the PPS of serotypes 3, 4, 6B, 9V, 14, and 23F and IgG antibodies to the pneumococcal virulence-associated proteins CbpA, LytC, PhtD and its C-terminal fragment (PhtD C), NanA, PspA fam1, and PspA fam2 were measured by enzyme immunoassay in the sera of younger (30 to 64 years of age) and elderly (65 to 97 years of age) adults. The concentrations of anti-PPS IgG against serotypes 3 and 6B, of anti-PPS IgM against serotypes 3, 4, 6B, 9V, and 23F, and of anti-protein IgG against all tested antigens were significantly lower in the elderly than in younger adults. A stronger decline in anti-PPS antibody concentrations was seen with age in women compared to men, while anti-protein antibody concentrations were mainly similar between the genders. Age, gender, and the nature of the antigen have substantial and varying effects on the antibody concentrations in the sera of adults.

Functional antibodies elicited by two heptavalent pneumococcal conjugate vaccines in the Finnish Otitis Media Vaccine Trial.

In the Finnish Otitis Media Vaccine Trial, the now-licensed pneumococcal conjugate vaccine containing polysaccharides conjugated to protein CRM(197) (PncCRM) and the experimental pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine (PncOMPC), showed similar efficacy profiles against acute otitis media despite different antibody concentrations in sera. We now report the opsonophagocytic activities (OPA) in these sera. OPA, antibody concentration, and avidity for serotypes 6B, 19F, and 23F were determined in sera of infants who received either pneumococcal conjugate (PCV) or control vaccine at 2, 4, and 6 months of age and either the homologous or pneumococcal polysaccharide vaccine at 12 months of age. OPA varied by vaccine and serotype. The majority of PCV recipients had positive OPA after the fourth dose, while OPA was undetectable in the control group. Coinciding with the efficacy data, the concentration of antibodies required for 50% killing was low for 6B and high for 19F for both PCVs. Contradictory to the efficacy data, PncOMPC induced lower functional capacity to 23F than PncCRM. OPA correlated with antibody concentration, while avidity and functional capacity of antibodies showed no correlation. The OPA data provide valuable additional information for serotype-specific differences in protection and when evaluating serotype-specific immunogenicity and should thus be considered when defining serological correlates of protection.

Antibody response to 7-valent conjugated pneumococcal vaccine in patients with chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a common adulthood mature B-cell neoplasm. Infections are the most important cause of mortality in this condition, and Streptococcus pneumoniae has been considered the most important single pathogen. We investigated the immunogenicity of 7-valent pneumococcal conjugate vaccine in patients with CLL. The study material comprised 52 patients with CLL and 25 age- and sex-matched controls. The subjects were vaccinated with Prevenar pneumococcal conjugate vaccine. Serum samples were taken for antibody determinations before and four weeks after vaccination. Antibody response rates to vaccine antigens were lower in patients with CLL compared to controls. However, if the vaccine had been administered at an early stage of the disease, i.e. before commencement of chemotherapy and the development of hypogammaglobulinaemia, a significant vaccination response to at least six antigens was obtained in almost 40% of the CLL patients. Our results indicate that early administration of conjugate vaccine may be beneficial in CLL.

Kinetics and avidity of antibodies evoked by heptavalent pneumococcal conjugate vaccines PncCRM and PncOMPC in the Finnish Otitis Media Vaccine Trial.

The licensure of new pneumococcal conjugate vaccines (PCVs) relies on immunogenicity data. When defining correlates of protection, vaccine efficacy data must be included. In the FinOM Vaccine Efficacy Trial, the PncOMPC vaccine showed an efficacy profile similar to that of the licensed PncCRM vaccine despite different antibody responses after primary and booster vaccinations. We determined antibody kinetics and avidities in a subgroup of infants participating in the FinOM trial. A total of 166 infants in three vaccine groups were immunized at 2, 4, 6, and 12 months of age with 7-valent PCV, PncCRM or PncOMPC, or hepatitis B vaccine. Concentrations of serum immunoglobulin G (IgG) against pneumococcal capsular polysaccharides were determined at 2, 4, 6, 7, 12, 13, and 24 months of age, and the avidity index (AI) to serotypes 6B, 19F, and 23F were determined at 7, 12, 13, and 24 months of age by enzyme immunoassay. Both PCVs were highly immunogenic, but they demonstrated different kinetics of antibody response; the concentration of IgG against serotypes 6B, 19F, and 23F declined faster after the third and fourth doses of vaccine in the PncCRM group than in the PncOMPC group. For both PCVs, the mean AI of anti-6B and -23F, but not of anti-19F, increased during the follow-up, which is in line with serotype-specific protection in the FinOM trial. Our data suggest that the kinetics and avidities of antibodies should be considered, in addition to antibody responses, when defining correlates of protection.

Haemophilus ducreyi targets Src family protein tyrosine kinases to inhibit phagocytic signaling.

Haemophilus ducreyi, the etiologic agent of the sexually transmitted disease chancroid, has been shown to inhibit phagocytosis of both itself and secondary targets in vitro. Immunodepletion of LspA proteins from H. ducreyi culture supernatant fluid abolished this inhibitory effect, indicating that the LspA proteins are necessary for the inhibition of phagocytosis by H. ducreyi. Fluorescence microscopy revealed that macrophages incubated with wild-type H. ducreyi, but not with a lspA1 lspA2 mutant, were unable to complete development of the phagocytic cup around immunoglobulin G-opsonized targets. Examination of the phosphotyrosine protein profiles of these two sets of macrophages showed that those incubated with wild-type H. ducreyi had greatly reduced phosphorylation levels of proteins in the 50-to-60-kDa range. Subsequent experiments revealed reductions in the catalytic activities of both Lyn and Hck, two members of the Src family of protein tyrosine kinases that are known to be involved in the proximal signaling steps of Fcgamma receptor-mediated phagocytosis. Additional experiments confirmed reductions in the levels of both active Lyn and active Hck in three different immune cell lines, but not in HeLa cells, exposed to wild-type H. ducreyi. This is the first example of a bacterial pathogen that suppresses Src family protein tyrosine kinase activity to subvert phagocytic signaling in hostcells.

Inhibition of phagocytosis by Haemophilus ducreyi requires expression of the LspA1 and LspA2 proteins.

Haemophilus ducreyi previously has been shown to inhibit the phagocytosis of both secondary targets and itself by certain cells in vitro. Wild-type H. ducreyi strain 35000HP contains two genes, lspA1 and lspA2, whose encoded protein products are predicted to be 456 and 543 kDa, respectively. An isogenic mutant of H. ducreyi 35000HP with inactivated lspA1 and lspA2 genes has been shown to exhibit substantially decreased virulence in the temperature-dependent rabbit model for chancroid. This lspA1 lspA2 mutant was tested for its ability to inhibit phagocytosis of immunoglobulin G-opsonized particles by differentiated HL-60 and U-937 cells and by J774A.1 cells. The wild-type strain H. ducreyi 35000HP readily inhibited phagocytosis, whereas the lspA1 lspA2 mutant was unable to inhibit phagocytosis. Similarly, the wild-type strain was resistant to phagocytosis, whereas the lspA1 lspA2 mutant was readily engulfed by phagocytes. This inhibitory effect of wild-type H. ducreyi on phagocytic activity was primarily associated with live bacterial cells but could also be found, under certain conditions, in concentrated H. ducreyi culture supernatant fluids that lacked detectable outer membrane fragments. Both the wild-type strain and the lspA1 lspA2 mutant attached to phagocytes at similar levels. These results indicate that the LspA1 and LspA2 proteins of H. ducreyi are involved, directly or indirectly, in the antiphagocytic activity of this pathogen, and they provide a possible explanation for the greatly reduced virulence of the lspA1 lspA2 mutant.

Mutations in the lspA1 and lspA2 genes of Haemophilus ducreyi affect the virulence of this pathogen in an animal model system.

Haemophilus ducreyi 35000HP contains two genes, lspA1 and lspA2, whose predicted protein products have molecular weights of 456,000 and 543,000, respectively (C. K. Ward, S. R. Lumbley, J. L. Latimer, L. D. Cope, and E. J. Hansen, J. Bacteriol. 180:6013-6022, 1998). We have constructed three H. ducreyi 35000HP mutants containing antibiotic resistance cartridges in one or both of the lspA1 and lspA2 open reading frames. Western blot analysis using LspA1- and LspA2-specific monoclonal antibodies indicated that the wild-type parent strain 35000HP expressed LspA1 protein that was readily detectable in culture supernatant fluid together with a barely detectable amount of LspA2 protein. The lspA2 mutant 35000HP.2 expressed LspA1 protein that was detectable in culture supernatant fluid and no LspA2 protein. In contrast, the H. ducreyi lspA1 mutant 35000HP.1, which did not express the LspA1 protein, expressed a greater quantity of the LspA2 protein than did the wild-type parent strain. The lspA1 lspA2 double mutant 35000HP.12 expressed neither LspA1 nor LspA2. The three mutant strains adhered to human foreskin fibroblasts and to a human keratinocyte cell line in vitro at a level that was not significantly different from that of the wild-type strain 35000HP. Lack of expression of the LspA1 protein by both the lspA1 mutant and the lspA1 lspA2 double mutant was associated with an increased tendency to autoagglutinate. When evaluated in the temperature-dependent rabbit model for chancroid, the lspA1 lspA2 double mutant was substantially less virulent than the wild-type strain 35000HP. The results of these studies indicated that H. ducreyi requires both the LspA1 and LspA2 proteins to be fully virulent in this animal model for experimental chancroid.

Haemophilus ducreyi requires an intact flp gene cluster for virulence in humans.

An intact Haemophilus ducreyi flp operon is essential for microcolony formation in vitro. tadA is the 9th of 15 genes in the operon and has homology to NTPases of type IV secretion systems. Fifteen human volunteers were experimentally infected with both H. ducreyi 35000HP and the tadA mutant, 35000HP.400. Papules developed at similar rates at sites inoculated with the mutant and parent, while pustules formed at 36.4% of parent sites and at 0% of mutant sites (P = 0.001). Compared to 35000HP, 35000HP.400 had only a modest but significant reduction in lesion scores in the temperature-dependent rabbit model of chancroid. These data suggest that proteins secreted by the flp locus are required for full expression of virulence by H. ducreyi in humans but have less of a role in virulence in an animal model of infection.