1H, 13C and 15N resonance assignment of 6aJL2(R25G), a highly fibrillogenic λVI light chain variable domain

An allotypic variation at position 25 influences the fibrillogenicity of λVI light chains, which are related to humoral immune response and have been associated with AL amyloidosis. The full resonance assignment and a preliminary structural characterization of 6aJL2(R25G) are reported. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Single Mutation in Framework 2 of the Heavy Variable Domain Improves the Properties of a Diabody and a Related Single-Chain Antibody

Excellent results regarding improved therapeutic properties have been often obtained through the conversion of a single‐chain variable fragment (scFv) into a noncovalent dimeric antibody (diabody) via peptide linker shortening. We utilized this approach to obtain a dimeric version of the human scFv 6009F, which was originally engineered to neutralize the Cn2 toxin of Centruroides noxius scorpion venom. However, some envenoming symptoms remained with diabody 6009F. Diabody 6009F was subjected to directed evolution to obtain a variant capable of eliminating envenoming symptoms. After two rounds of biopanning, diabody D4 was isolated. It exhibited a single mutation (E43G) in framework 2 of the heavy‐chain variable domain. Diabody D4 displayed an increase in T_m (thermal transition midpoint temperature) of 6.3 °C compared with its dimeric precursor. The importance of the E43G mutation was tested in the context of the human scFv LR, a highly efficient antibody against Cn2, which was previously generated by our group [Riaño-Umbarila, L., Contreras-Ferrat, G., Olamendi-Portugal, T., Morelos-Juárez, C., Corzo, G., Possani, L. D. and Becerril, B. (2011). J. Biol. Chem. 286, 6143–6151]. The new variant, scFv LER, displayed an increase in T_m of 3.4 °C and was capable of neutralizing 2 LD₅₀ of Cn2 toxin with no detectable symptoms when injected into mice at a 1:1 toxin-to-antibody molar ratio. These results showed that the E43G mutation might increase the therapeutic properties of these antibody fragments. Molecular modeling and dynamics results suggest that the rearrangement of the hydrogen-bonding network near the E43G mutation could explain the improved functional stability and neutralization properties of both the diabody D4 and scFv LER.     

Evolutionary Dynamics of ompA,the Gene Encoding the Chlamydia trachomatis Key Antigen

Chlamydia trachomatis is the trachoma agent and causes most bacterial sexually transmitted infections worldwide. Its major outer membrane protein (MOMP) is a well-known porin and adhesin and is the dominant antigen. So far, investigation of MOMP variability has been focused mainly on molecular epidemiological surveys. In contrast, we aimed to evaluate the impact of the host pressure on this key antigen by analyzing its evolutionary dynamics in 795 isolates from urogenital infections, taking into account the MOMP secondary structure and the sizes/positions of antigenic regions. One-third of the specimens showed a mutational drift from the corresponding genotype, where ∼42% of the mutations had never been described. Amino acid alterations were sixfold more frequent within B-cell epitopes than in the remaining protein (P = 0.027), and some mutations were also found within or close to T-cell antigenic clusters. Interestingly, the two most ecologically successful genotypes, E and F, showed a mutation rate 60.3-fold lower than that of the other genotypes (P < 10⁻⁸), suggesting that their efficacy may be the result of a better fitness in dealing with the host immune system rather than of specific virulence factors. Furthermore, the variability exhibited by some genetic variants involved residues that are known to play a critical role during the membrane mechanical movements, contributing to a more stable and flexible porin conformation, which suggests some plasticity to deal with environmental pressure. Globally, these MOMP mutational trends yielded no mosaic structures or important phylogenetic changes, but instead yielded point mutations on specific protein domains, which may enhance pathogen''s infectivity, persistence, and transmission.Chlamydia trachomatis is an obligate intracellular human pathogen that can be classified into 18 serovariants based on the immunoreactivity of its major outer membrane protein (MOMP) or ompA (which encodes MOMP) polymorphism: serovars A to C and Ba are responsible for trachoma; serovars D to K, Da, Ia, and Ja are normally associated with infection of the urogenital tract; and serovars L1 to L3 cause lymphogranuloma venereum (22). This preference for particular cell types is not exclusive, and therefore ocular strains can occasionally be found in the urogenital tract and vice versa. However, it is thought that only L1 to L3 strains possess the ability to invade the inguinal lymph nodes.MOMP has been implicated in the mechanisms of attachment, infection, and/or pathogenesis due to its variability, surface exposure, and antigenic properties. Previous studies have shown that MOMP may act as a putative cytoadhesin by promoting nonspecific interactions with host cells (64). This major chlamydial membrane component, which constitutes about 60% of the membrane dry weight (9), is also thought to play a role in maintaining structural integrity of the organism (9, 10) by forming a trimeric structure (66). Also, during chlamydial replication, MOMP may act as a porin (6) that is folded into a β-barrel structure containing five constant domains (CDI to CDV) of transmembrane β-strands and periplasmic turns and four highly variable surface-exposed domains (VDI to VDIV) (34, 59, 69). Furthermore, MOMP possesses species- and serovar-specific epitopes (2, 48, 73, 74) that are able to elicit both the humoral (B-cell mediated through the production of antibodies) and cellular (T-cell mediated and also influencing the B-cell response) immune responses, making this dominant chlamydial antigen a potential candidate for the development of vaccines and therapeutic strategies (8, 17, 23, 61). Indeed, although no efficacious chlamydial vaccine has been developed so far, the use of inactivated or live-attenuated pathogens has been replaced by peptide or subunit vaccines, and MOMP is definitely one of the leading candidates (19).To improve our knowledge of the effects of the host pressure on MOMP and also of the molecular epidemiology of the circulating C. trachomatis strains, it is imperative to investigate genetic variability in ompA. Here, we performed a sequence-based analysis of the ompA mutational trends in clinical isolates that were collected from patients with sexually transmitted C. trachomatis infections. So far, most studies have been limited to a small number of strains with variations in ompA (16, 33, 36, 42, 44, 47, 49, 52, 57, 62) or were restricted to the analysis of VDs (7, 14, 15, 32, 63), discarding the CDs, which contain numerous cytotoxic T lymphocyte (CTL) and T helper (Th) cell epitopes (30, 38, 39, 55, 56). We performed a detailed bioinformatic and statistical analysis of the mutational dispersion on both VDs and CDs, based on MOMP structure and on the mapping of all the B- and T-cell epitopes reported in the literature. We present statistically validated genomic evidence of the adaptation of this pathogen''s key antigen to the host pressure, which strongly indicates a strategy to evade the human immune system.     

Influence of the protein structure surrounding the active site on the catalytic activity of [NiFeSe] hydrogenases

A combined experimental and theoretical study of the catalytic activity of a [NiFeSe] hydrogenase has been performed by H/D exchange mass spectrometry and molecular dynamics simulations. Hydrogenases are enzymes that catalyze the heterolytic cleavage or production of H₂. The [NiFeSe] hydrogenases belong to a subgroup of the [NiFe] enzymes in which a selenocysteine is a ligand of the nickel atom in the active site instead of cysteine. The aim of this research is to determine how much the specific catalytic properties of this hydrogenase are influenced by the replacement of a sulfur by selenium in the coordination of the bimetallic active site versus the changes in the protein structure surrounding the active site. The pH dependence of the D₂/H⁺ exchange activity and the high isotope effect observed in the Michaelis constant for the dihydrogen substrate and in the single exchange/double exchange ratio suggest that a “cage effect” due to the protein structure surrounding the active site is modulating the enzymatic catalysis. This “cage effect” is supported by molecular dynamics simulations of the diffusion of H₂ and D₂ from the outside to the inside of the protein, which show different accumulation of these substrates in a cavity next to the active site.     

Estimates of pandemic influenza vaccine effectiveness in Europe, 2009-2010: results of Influenza Monitoring Vaccine Effectiveness in Europe (I-MOVE) multicentre case-control study

Background

A multicentre case-control study based on sentinel practitioner surveillance networks from seven European countries was undertaken to estimate the effectiveness of 2009–2010 pandemic and seasonal influenza vaccines against medically attended influenza-like illness (ILI) laboratory-confirmed as pandemic influenza A (H1N1) (pH1N1).

Methods and Findings

Sentinel practitioners swabbed ILI patients using systematic sampling. We included in the study patients meeting the European ILI case definition with onset of symptoms >14 days after the start of national pandemic vaccination campaigns. We compared pH1N1 cases to influenza laboratory-negative controls. A valid vaccination corresponded to >14 days between receiving a dose of vaccine and symptom onset. We estimated pooled vaccine effectiveness (VE) as 1 minus the odds ratio with the study site as a fixed effect. Using logistic regression, we adjusted VE for potential confounding factors (age group, sex, month of onset, chronic diseases and related hospitalizations, smoking history, seasonal influenza vaccinations, practitioner visits in previous year). We conducted a complete case analysis excluding individuals with missing values and a multiple multivariate imputation to estimate missing values. The multivariate imputation (n = 2902) adjusted pandemic VE (PIVE) estimates were 71.9% (95% confidence interval [CI] 45.6–85.5) overall; 78.4% (95% CI 54.4–89.8) in patients <65 years; and 72.9% (95% CI 39.8–87.8) in individuals without chronic disease. The complete case (n = 1,502) adjusted PIVE were 66.0% (95% CI 23.9–84.8), 71.3% (95% CI 29.1–88.4), and 70.2% (95% CI 19.4–89.0), respectively. The adjusted PIVE was 66.0% (95% CI −69.9 to 93.2) if vaccinated 8–14 days before ILI onset. The adjusted 2009–2010 seasonal influenza VE was 9.9% (95% CI −65.2 to 50.9).

Conclusions

Our results suggest good protection of the pandemic monovalent vaccine against medically attended pH1N1 and no effect of the 2009–2010 seasonal influenza vaccine. However, the late availability of the pandemic vaccine and subsequent limited coverage with this vaccine hampered our ability to study vaccine benefits during the outbreak period. Future studies should include estimation of the effectiveness of the new trivalent vaccine in the upcoming 2010–2011 season, when vaccination will occur before the influenza season starts. Please see later in the article for the Editors'' Summary