Evaluation of glycoconjugate antigens as vaccine candidates against group A Streptococcus and human immunodeficiency virus infections

This PhD thesis discusses the rationale for design and use of synthetic oligosaccharides for the development of glycoconjugate vaccines and the role of physicochemical methods in the characterization of these vaccines. The study concerns two infectious diseases that represent a serious problem for the national healthcare programs: human immunodeficiency virus (HIV) and Group A Streptococcus (GAS) infections. Both pathogens possess distinctive carbohydrate structures that have been described as suitable targets for the vaccine design. The Group A Streptococcus cell membrane polysaccharide (GAS-PS) is an attractive vaccine antigen candidate based on its conserved, constant expression pattern and the ability to confer immunoprotection in a relevant mouse model. Analysis of the immunogenic response within at-risk populations suggests an inverse correlation between high anti-GAS-PS antibody titres and GAS infection cases. Recent studies show that a chemically synthesized core polysaccharide-based antigen may represent an antigenic structural determinant of the large polysaccharide. Based on GAS-PS structural analysis, the study evaluates the potential to exploit a synthetic design approach to GAS vaccine development and compares the efficiency of synthetic antigens with the long isolated GAS polysaccharide. Synthetic GAS-PS structural analogues were specifically designed and generated to explore the impact of antigen length and terminal residue composition. For the HIV-1 glycoantigens, the dense glycan shield on the surface of the envelope protein gp120 was chosen as a target. This shield masks conserved protein epitopes and facilitates virus spread via binding to glycan receptors on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope has been a subject to the synthetic vaccine development. The cluster nature of the 2G12 epitope suggested that multivalent antigen presentation was important to develop a carbohydrate based vaccine candidate. I describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates and their immunogenic properties.

Studio immunologico per l'individuazione di pazienti trapiantati per cirrosi HBV relata che potrebbero sospedere la terapia con immunoglobine specifiche (HBIG)

Hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT) is associated with poor graft and patient survival. Treatment with HBV-specific immunoglobulins (HBIG) in combination with nucleos(t)ide analogs is effective in preventing HBV reinfection of the graft and improving OLT outcome. However, the combined immunoprophylaxis has several limitations, mainly the high cost and the lack of standard schedules about duration. So far, the identification of markers able to predict the reinfection risk is needed. Although the HBV-specific immune response is believed to play an essential role in disease outcome, HBV-specific cellular immunity in viral containment in OLT recipients is unclear. To test whether or not OLT recipients maintain robust HBV-specific cellular immunity, the cellular immune response against viral nucleocapsid and envelope-protein of HBV was assessed in 15 OLT recipients and 27 individuals with chronic and 24 subjects with self-limited HBV infection, respectively. The data demonstrate that OLT recipients mounted fewer but stronger clusters of differentiation (CD)8 T cell responses than subjects with self-limited HBV infection and showed a preferential targeting of the nucleocapsid antigen. This focused response pattern was similar to responses seen in chronically infected subjects with undetectable viremia, but significantly different from patients who presented with elevated HBV viremia and who mounted mainly immune responses against the envelope protein. In conclusion, virus-specific CD4 T cell–mediated responses were only detected in subjects with self-limited HBV infection. Thus, the profile of the cellular immunity against HBV was in immune suppressed patients similar to subjects with chronic HBV infection with suppressed HBV-DNA.

Identificazione delle Lamine di tipo A come nuovi substrati della protein chinasi Akt/PKB

Akt (also called PKB) is a 63 kDa serine/threonine kinase involved in promotion of cell survival, proliferation a nd metabolic responses downstream the phosphoinositide-3-kinase (PI 3-kinase) signaling pathway. In resting cells, Akt is a predominantly cytosolic enzyme; however generation of PI 3-kinase lipid products recruits Akt to the plasma membrane, resulting in a conformational change which confers full enzymatic activity through the phosphorylation of the membrane-bound protein at two residues, Thr308, and Ser473. Activated Akt redistributes to cytoplasm and nucleus, where phosphorylation of specific substrates occurs. Both the presence and the activity of Akt in the nucleus have been described. An interesting mechanism that mediates nuclear translocation of Akt has been described in human mature T-cell leukemia: the product of TCL1 gene, Tcl1, interacts with the PH domain of phosphorylated Akt, thus driving Akt to the nucleus. In this context, Tcl1 may act as a direct transporter of Akt or may contribute to the formation of a complex that promotes the transport of active Akt to the nucleus, where it can phosphorylate nuclear substrates. A well described nuclear substrate if Foxo. IGF-1 triggers phosphorylation of Foxo by Akt inside the nucleus, where phospho-Foxo associates to 14.3.3 proteins that, in turn, promote its export to the cytoplasm where it is sequestered. Remarkably, Foxo phosphorylation by Akt has been shown to be a crucial event in Akt-dependent myogenesis. However, most Akt nuclear substrates have so far remained elusive, as well as nuclear Akt functions. This lack of information prompted us to undertake a search of substrates of Akt in the nucleus, by the combined use of 2D-separation/mass spectrometry and anti-Akt-phosphosubstrate antibody. This study presents evidence of A-type lamins as novel nuclear substrates of Akt. Lamins are type V intermediate filaments proteins found in the nucleus of higher eukaryotes where, together with lamin-binding proteins, they form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane. By coimmunoprecipitation, it is demonstrated here that endogenous lamin A and Akt interact, and that A-type lamins are phosphorylated by Akt both in vitro and in vivo. Moreover, by phosphoaminoacid analysis and mutagenesis, it is further demonstrated that Akt phosphorylates lamin A at Ser404, and, more importantly, that while lamin A/C phosphorylation is stable throughout the cell cycle, phosphorylation of the precursor prelamin A becomes detectable as cells enter the G2 phase, picking at G2/M. This study also shows that lamin phosphorylation by Akt creates a binding site for 14.3.3 adaptors which, in turn, promote prelamin A degradation. While this mechanism is in agreement with a general role of Akt in the regulation of a subset of its substrates, opposite to what has been described, degradation is not mediated through a ubiquitination and proteasomal mechanism but through a lysosomal pathway, as indicated by the reverting action of the lysosomal inhibitor cloroquine. Phosphorylation is a key event in the mitotic breakdown of the nuclear lamina. However, the kinases and the precise sites of phosphorylation are scarcely known. Therefore, these results represent an important breakthrough in this very significant but understudied area. The phosphorylation of the precursor protein prelamin A and its subsequent degradation at G2/M, when both the nuclear envelop and the nuclear lamina disassemble, can be view as part of a mechanism to dispose off the precursor that is not needed in this precise context. The recently reported finding that patients affected by Emery-Dreifuss muscular dystrophy carry a mutation at Arg 401, in the Akt phosphorylation motif, open new perspective that warrant further investigation in this very important field.

Analisi dei meccanismi patogenetici alla base del danno vascolare Hiv-indotto

L'infezione da HIV-1 resta ancora oggi una delle principali problematiche nell'ambito della sanità mondiale, con circa 35 milioni di individui infetti in tutto il mondo. L'introduzione della terapia antiretrovirale combinata (cART) ha drasticamente modificato l’evoluzione di questa infezione, che da patologia a sviluppo terminale dopo alcuni anni dalla trasmissione, è diventata una patologia cronica con una lunga aspettativa di vita per i pazienti. Tuttavia, la cART non è in grado di eradicare l’infezione e nei pazienti HIV-infetti trattati è possibile notare un aumento della comparsa di comorbidità, tra le quali le più frequentemente riscontrate sono lesioni al sistema nervoso centrale, ai reni, al tessuto osseo, al fegato e al sistema cardiovascolare. I danni al sistema cardiocircolatorio derivano da una serie di concause virologiche, comportamentali, ambientali e farmacologiche che alterano la parete vascolare, il metabolismo dei lipidi e la regolazione della coagulazione, inducendo la formazione di lesioni strutturali di tipo aterosclerotico che sono alla base dell’aumentata incidenza di infarti, ictus e alterazioni del circolo osservabili nei pazienti HIV-positivi. Dalla recente letteratura è emerso come l’omeostasi del tessuto endoteliale sia regolata anche a livello delle cellule staminali mesenchimali (MSC) presenti nella parete vascolare. Per questo abbiamo voluto analizzare possibili effetti dell’infezione di HIV, delle sue proteine e di alcune molecole antiretrovirali sulla vitalità e sul differenziamento delle MSC purificate dalla parete arteriosa umana. I risultati ottenuti indicano come l’infezione da HIV e l’azione delle proteine gp120 e Tat attivino il meccanismo di apoptosi nelle MSC e una profonda alterazione nel differenziamento verso la filiera adipocitaria e verso quella endoteliale. Inoltre, alcune molecole ad azione antiretrovirale (in particolare specifici inibitori della proteasi virale) sono in grado bloccare il differenziamento delle MSC verso le cellule endoteliali. Dall’insieme di queste osservazioni emergono nuovi meccanismi patogenetici correlati al danno cardiovascolare riscontrato nei pazienti HIV-positivi.

Studio dell'interazione di HIV-1 sulle cellule progenitrici ematopoietiche CD34+ (HPCs)

Oltre alla progressiva perdita dei linfociti T CD4, i pazienti HIV-infetti presentano diverse citopenie periferiche. In particolare l’anemia si riscontra nel 10% dei pazienti asintomatici e nel 92% di quelli con AIDS e la terapia cART non è in grado di risolvere tale problematica. I meccanismi patogenetici alla base di questa citopenia si ritiene che possano riguardare la deregolazione citochinica, il danno alle HPCs, alle cellule in differenziamento e alle cellule stromali. Le cellule progenitrici ematopoietiche CD34+, dopo essere state separate da sangue cordonale e differenziate verso la linea eritroide, sono state trattate con HIV-1 attivo, inattivato al calore e gp120. In prima istanza è stata messa in luce la mancata suscettibilità all’infezione e l’aumento dell’ apoptosi dovuto al legame gp120-CD4/CXCR4 e mediato dal TGF-β1 nelle cellule progenitrici indifferenziate. L’aspetto innovativo di questo studio però si evidenzia esaminando l’effetto di gp120 durante il differenziamento verso la filiera eritrocitaria. Sono stati utilizzati due protocolli sperimentali: nel primo le cellule sono inizialmente trattate per 24 ore con gp120 (o con HIV-1 inattivato al calore) e poi indotte in differenziamento, nel secondo vengono prima differenziate e poi trattate con gp120. Il “priming” negativo determina una apoptosi gp120-indotta molto marcata già dopo 48 ore dal trattamento ed una riduzione del differenziamento. Se tali cellule vengono invece prima differenziate per 24 ore e poi trattate con gp120, nei primi 5 giorni dal trattamento, è presente un aumento di proliferazione e differenziamento, a cui segue un brusco arresto che culmina con una apoptosi molto marcata (anch’essa dipendente dal legame gp120-CD4 e CXCR4 e TGF-β1 dipendente) e con una drastica riduzione del differenziamento. L’insieme dei risultati ha permesso di definire in modo consistente la complessità della genesi dell’anemia in questi pazienti e di poter suggerire nuovi target terapeutici in questi soggetti, già sottoposti a cART.