Crystal Structure of the Neutralizing Llama VHH D7 and Its Mode of HIV-1 gp120 Interaction

HIV-1 entry into host cells is mediated by the sequential binding of the envelope glycoprotein gp120 to CD4 and a chemokine receptor. Antibodies binding to epitopes overlapping the CD4-binding site on gp120 are potent inhibitors of HIV entry, such as the llama heavy chain antibody fragment VHH D7, which has cross-clade neutralizing properties and competes with CD4 and mAb b12 for high affinity binding to gp120. We report the crystal structure of the D7 VHH at 1.5 Å resolution, which reveals the molecular details of the complementarity determining regions (CDR) and substantial flexibility of CDR3 that could facilitate an induced fit interaction with gp120. Structural comparison of CDRs from other CD4 binding site antibodies suggests diverse modes of interaction. Mutational analysis identified CDR3 as a key component of gp120 interaction as determined by surface plasmon resonance. A decrease in affinity is directly coupled to the neutralization efficiency since mutations that decrease gp120 interaction increase the IC50 required for HIV-1 IIIB neutralization. Thus the structural study identifies the long CDR3 of D7 as the key determinant of interaction and HIV-1 neutralization. Furthermore, our data confirm that the structural plasticity of gp120 can accommodate multiple modes of antibody binding within the CD4 binding site

Llama Antibody Fragments Recognizing Various Epitopes of the CD4bs Neutralize a Broad Range of HIV-1 Subtypes A, B and C

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120Ds2), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides

Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols

Development of llama single-domain antibodies as ingredient for an HIV -1 entry-inhibitor microbicide

Heavy-chain antibodies are present in llamas next to conventional immunoglobulins. Their variable domain, also known as VHH is a small, single-chain globular protein with full antigen-binding capacity. Its single-domain nature and some structural differences from conventional immunoglobulins make it a tool with high potential for a number of biotechnological and medical applications. The HIV pandemic represents since three decades a dramatic health problem throughout the world and especially in resource-limited countries. As an alternative to vaccination, prevention is regarded as the best way to control the pandemic, both from a healthcare and economic point of view. The development of microbicides that help preventing sexually transmitted infections of HIV (the most common way the virus is transmitted) is therefore highly desirable. In this thesis, several aspects of the development of llama heavy-chain antibody fragments as active compound of topical microbicides are investigated. In chapter one the 3D structure of the HIV neutralizing VHH D7 was determined. The extension, flexibility and amino acid-composition of its CDRs were compared to other known (human) neutralizing antibodies in order to define the way D7 neutralizes the virus by binding gp120. Structural comparison suggested diverse modes of interaction. Mutational analysis identified CDR3 as key area of the interaction with gp120, whose conformational flexibility is likely to accommodate multiple modes of antibody binding inside the CD4 binding site. Comparison of the related VHH A12 and D7 revealed broad and narrow cross-clade neutralization phenotypes respectively. In chapter two a family-specific phage library was generated and a number of homologous VHH was selected with varying neutralizing profiles. Analysis of their sequences and their recognition of a panel of different HIV-1 subtypes, allowed the understanding of the molecular basis of the different neutralization behaviors. In particular the important role played by the last two C-terminal amino acids of CDR3 was demonstrated. Secretion efficiency of VHH is a particularly important factor when large amounts are needed at low production cost. In chapter three we analyzed databases of structures and sequences to find sequence-related factors that influence the yield of VHH when produced in the host S. cerevisiae. We showed how the combined presence of five key residues and specific J segments is fundamental for proper folding. We also suggested that interactions with ER-resident chaperones are necessary to achieve high secretion efficiency. In the last chapter, VHH A12 and D7 were used as models to study their availability, activity and stability in a VHH-based topical microbicide. We systematically analyzed likely challenges that VHH will have to face in case of use in sub-tropical settings of resource-limited countries, as HIV preventative drug. VHH proved to be readily available when formulated as tablets or aqueous gel and very stable after extended incubation at physiological temperature and at low pH. We also investigated the tissue-penetration kinetics of VHH in order to assess whether the drug would be present in the sub-mucosal layers of the vagina and once there, carry out its protective action

Quale rilancio per le autonomie territoriali?

Il presente volume raccoglie gli Atti del Convegno dall’analogo titolo svoltosi presso l’Università di Brescia in data 24 maggio 2019. La riflessione che si è intesa proporre attraverso il Convegno, prima, ed il volume collettaneo, poi, ha preso le mosse dalla crisi in cui da tempo versa il sistema delle autonomie territoriali. Le cause, plurime quanto note, sono di natura legislativa, amministrativa e finanziaria e, più in generale, di politica istituzionale, al punto che lo stesso dibattito dottrinale ne ha risentito entrando in una sorta di “stallo”. Da qui l’esigenza di ri-lanciare la discussione dando vita ad un ripensamento e forse anche ad un riposizionamento culturale sul tema. Gli interventi degli Autori, nonché rimettere ordine in una complessa quanto articolata riflessione scientifica e politica, oltre che tener conto delle recenti richieste di autonomia differenziata ex art. 116, c. 3, Cost. avanzate da alcune regioni e dello stato di “congelamento” delle autonomie provinciali, giungono infine, in prospettiva de jure condendo, ad approfondire la tematica delle possibili riforme di natura costituzionale e legislativa

Allevamento e macellazione del suino pesante: correlazione tra la qualit\ue0 della carne e il grado di benessere (Correlation between meat quality and welfare level: the case of italian heavy pigs)

Scopo della presente ricerca \ue8 stato quello d\u2019indagare le relazioni esistenti fra il benessere degli animali durante le fasi di allevamento e trasporto e la qualit\ue0 delle carni da essi ottenute. La sperimentazione \ue8 stata condotta su 44 partite di suini pesanti provenienti da 11 allevamenti siti in Emilia Romagna e selezionati casualmente. Il benessere animale \ue8 stato valutato dal Centro Ricerche e Produzioni Animali (CRPA) di Reggio-Emilia attraverso un sistema da loro implementato: per ogni allevamento \ue8 stato calcolato l\u2019Indice di Benessere dell\u2019Allevamento (IBA) e per ogni partita di macellazione l\u2019Indice di Benessere del Trasporto e della pre-Macellazione (IBTM). Da 440 animali (10 per partita) \ue8 stato prelevato un campione di sangue alla giugulazione su cui sono state determinate le concentrazioni di creatinchinasi (CK), aldolasi e cortisolo. Dai medesimi animali sono stati poi prelevati campioni di lombo su cui sono stati misurati pH, colore strumentale, potere di ritenzione idrica e resistenza al taglio. I risultati dell\u2019analisi statistica hanno evidenziato che il miglioramento del benessere influenza in positivo la qualit\ue0 della carne: in tal senso l\u2019effetto pi\uf9 importante non \ue8 dovuto alle condizioni d\u2019allevamento, bens\uec a quelle di trasporto e macellazione. Nello specifico i punti critici individuati dal nostro studio sono rappresentati dal prolungamento del tempo di trasporto e dalla commistione di animali provenienti da gruppi sociali diversi.The present study investigated the relationships between the welfare of pigs (on farm, during transport and before slaughtering) and the quality of the derived meat. The experimental trial was carried out on 44 slaughtering batches of heavy pigs, coming from 11 farms located in Emilia Romagna and randomly selected. Animal welfare was assessed by the Research Centre on Animal Production (Italian initials CRPA) of Reggio Emilia, according to a welfare assessment protocol they have implemented. According to this protocol, each farm was scored for the Farm Welfare Index (Italian initials IBA), and each slaughtering batch was scored for the Transport and pre-Slaughter Welfare Index (Italian initials IBTM). Blood samples were collected upon exsanguination from 440 pigs (10 per slaughtering batch). Cortisol, creatinkinase (CK) and aldolase concentrations were analyzed on plasma. Loin samples were collected from the same animals, and used for meat quality evaluations (pH, instrumental colour, water holding capacity, shear force). The results of the statistical analysis underlined that an increase in welfare level had a positive effect on meat quality; transport and pre-slaughtering procedures had the most important effects, whereas the level of welfare on farm had a lower effect. The critical points observed in this study were represented by the increase in journey duration and the mixing of pigs from different social groups during transport and lairage