Computational design of protein antigens that interact with the CDR H3 loop of HIV broadly neutralizing antibody 2F5.

Rational design of proteins with novel binding specificities and increased affinity is one of the major goals of computational protein design. Epitope-scaffolds are a new class of antigens engineered by transplanting viral epitopes of predefined structure to protein scaffolds, or by building protein scaffolds around such epitopes. Epitope-scaffolds are of interest as vaccine components to attempt to elicit neutralizing antibodies targeting the specified epitope. In this study we developed a new computational protocol, MultiGraft Interface, that transplants epitopes but also designs additional scaffold features outside the epitope to enhance antibody-binding specificity and potentially influence the specificity of elicited antibodies. We employed MultiGraft Interface to engineer novel epitope-scaffolds that display the known epitope of human immunodeficiency virus 1 (HIV-1) neutralizing antibody 2F5 and that also interact with the functionally important CDR H3 antibody loop. MultiGraft Interface generated an epitope-scaffold that bound 2F5 with subnanomolar affinity (K(D) = 400 pM) and that interacted with the antibody CDR H3 loop through computationally designed contacts. Substantial structural modifications were necessary to engineer this antigen, with the 2F5 epitope replacing a helix in the native scaffold and with 15% of the native scaffold sequence being modified in the design stage. This epitope-scaffold represents a successful example of rational protein backbone engineering and protein-protein interface design and could prove useful in the field of HIV vaccine design. MultiGraft Interface can be generally applied to engineer novel binding partners with altered specificity and optimized affinity.

Direct antibody access to the HIV-1 membrane-proximal external region positively correlates with neutralization sensitivity.

On the prereceptor-engaged HIV-1 envelope glycoprotein (Env) spike, epitope access by the membrane-proximal external region (MPER)-directed broadly neutralizing antibodies 2F5 and 4E10 remains unresolved. Data on binding to cell surface Env and entry data using primary isolates suggest inaccessibility of the 2F5 and 4E10 epitopes on the viral spike prior to receptor engagement, but trimer gel shift analysis and slow kinetics of shedding induced by 2F5 and 4E10 indicate otherwise. Therefore, it remains unclear if the epitopes themselves are formed in their antibody-bound state (or at least sampled) prior to receptor/coreceptor engagement or if receptor interactions both expose and form the MPER epitopes, presumably in the putative prefusion transitional intermediate. Here, we performed antibody-virus "washout experiments" using both lab-adapted and a panel of clade B primary isolates to analyze MPER accessibility. The neutralization activity of 2F5 and 4E10 against lab-adapted viruses and sensitive and moderately resistant viruses was largely unaffected by relatively rapid antibody-virus washing, suggesting direct interaction with the "static" spike. However, for more neutralization-resistant viruses, the 2F5 and 4E10 antibodies could neutralize only under the "no antibody-virus wash" conditions, implying that the MPER epitopes were not accessible prior to receptor engagement. Accessibility in the washout conditions could be precisely predicted by the relative resistance to neutralization in a standard neutralization format. These data are consistent with a model in which the local MPER antibody epitope conformations may be sampled on the native spike but are occluded to antibody by local steric or distal quaternary constraints adopted by highly resistant HIV-1 isolates.

Effect of patient characteristics on reported outcomes after total knee replacement.

OBJECTIVE: To evaluate the effect of pre-intervention factors in patient-reported outcomes at 6 months post-operatively following total knee replacement. METHODS: A prospective observational study was carried out using two questionnaires sent to patients while they were on the waiting list for surgery: a generic questionnaire, the Medical Outcomes Study Short Form-36 (SF-36), and a specific questionnaire, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Six months after intervention, patients again received the same questionnaires. The dependent variables were the scores of the three domains of the WOMAC and the eight domains of the SF-36. RESULTS: We recruited 640 patients. The mean age was 71 yrs and 73.6% of the patients were females. The multivariate analysis, in which the pre-intervention scores for each domain were added as covariates, showed that the most significant pre-intervention predictors were the baseline scores of each domain. Besides that, the social support, low back pain and the baseline score of the mental health domain (SF-36) were the pre-intervention predictors in the three WOMAC domains. With regard to the SF-36 domains the main predictors were the baseline mental health score, comorbidities, low back pain and social support. CONCLUSIONS: The main predictor of outcome at 6 months post-operatively in all eleven domains was the pre-intervention score of each domain. Presence of social support, absence of low back pain and higher baseline SF-36 mental health score were related to the improvement in the health-related quality of life post-operatively.

Validation of the Spanish version of the WOMAC questionnaire for patients with hip or knee osteoarthritis. Western Ontario and McMaster Universities Osteoarthritis Index.

The aim of this study was to validate a translated version of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaire in Spanish patients with hip or knee osteoarthritis (OA). The WOMAC questionnaire and the SF-36 were administered to a sample of 269 patients on the waiting list for hip or knee replacement. We studied the convergent validity and the item-scale correlation using Pearson's correlation coefficient and Spearman's pi. For the reliability study we used another sample of 58 patients who received the WOMAC twice within 15 days. The Pearson's, Spearman's pi, and intraclass correlation coefficients were calculated. Internal consistency was measured by Cronbach's alpha. The responsiveness study was carried out by resending the two questionnaires to all patients 6 months after surgical intervention; responsiveness was measured by means of the paired t-test, the effect size I and the standardised response mean. The Pearson's coefficients for the convergent validity ranged from -0.52 to -0.63. The coefficients obtained for the item-scale correlation of the pain area were 0.74 or higher, 0.91 or higher for stiffness, and 0.61 or higher for function. When measuring the test-retest reliability, the coefficients ranged from 0.66 to 0.81. Internal consistency yielded a Cronbach's alpha ranging from 0.81 to 0.93. The responsiveness showed an effect size I ranging from 1.5 to 2.2 in patients who underwent hip replacement; for those who underwent knee replacement the range was 1 to 1.8. The standardised response mean ranged from 1.3 to 1.9 for patients with hip OA; those with knee OA ranged from 0.8 to 1.5. The Spanish version of WOMAC is a valid, reliable and responsive instrument in patients with hip or knee OA.

[Appropriate use of the mechanisms for the fixation of hip prostheses]. / Uso apropiado de los mecanismos de fijación en la prótesis de cadera.

OBJECTIVE: To evaluate the appropriateness of the use of fixation mechanisms for hip replacement in patients with osteoarthritis or prosthesis revision, using explicit criteria. METHODS: Following the RAND appropriateness method, it was carried out a revision of the bibliography and was created a list of 12 indications for osteoarthritis and 32 for the revision. A panel of 27 experts, all of them traumatologists, punctuated the degree of appropriateness for each indication in a scale of 1 (completely inappropriate) up to 9 (completely appropriate). According to the median of the punctuations and the agreement level, each indication was classified as appropriate, uncertain or inappropriate. The data of ten public hospitals of medium and big size of the Basque Service of Health-Osakidetza, were picked up and the developed criteria were applied to the evaluated interventions. RESULTS: 831 osteoarthritis interventions and 349 revisions were evaluated. Of the 136 interventions in which the prosthesis used was cemented, in two thirds its use was considered appropriate. The cementless prosthesis was used more frequently (77%) and in most of the cases (94%) it was considered appropriate according to the criteria of the panel. Of the 68 acetabular revisions in which cemented prosthesis was used, in 85% of them the use of cementless mechanism was recommended. However, of the 134 interventions that used cementless, the panel agreed on 94%. Similar results happened with the femoral revision. CONCLUSIONS: The study offers some recommendations on the use of the cemented and cementless prosthesis, based on the work of a panel of experts. Once applied, the criteria of the panel a certain level of uncertain use is observed.