FATTORI UMORALI PROTETTIVI COME STRUMENTI PER LA GENERAZIONE DI VACCINI EFFICACI CONTRO L'INFEZIONE DA HIV
Creators: name: DIOMEDE, LORENZO
Publisher: name: Universit� degli Studi di Milano
Year: 2012

description: The present study includes several works which have common goal to further clarify the humoral correlates of protection against HIV infection, present in those populations that naturally can remain IgG-seronegative, despite a long-term exposure to HIV (ESNs), or are able to control for a long time the same infection (LTNPs, ECs). In fact, the first part of the thesis is concerned about the study of protective humoral parameters which would characterize a cohort of 45 HIV-infected individuals at different clinical stages (10 AI, Acute Infection, 10 LTNPs, 8 ECs, 7 AIDS, 10 HAART +); a cohort consisting of South African mothers infected with HIV subtype C, coupled with their infants, some of which are seronegative (as a particular ESN group), and finally, a cohort of women in Cambodian ESN, whose B cells, in their cervicovaginal secretions, were used to construct a library of Fab IgA phage-k/?, which was screened for specificity to HIV-gp41. The results showed that heterogeneous humoral responses were observed in groups of HIV seropositive subjects. The high variability of total or gp41-specific IgA among all patients groups, supported their role as a biomarker. Infact, significant differences were detected in AI and in LTNP for IgA to gp41 and in IgG to gp120, respectively. Moreover, three domains of gp41, HR1, IDE and MPER, elicited antibodies that were effectively transmitted to ESN babies. In such babies, epitopes overlapping the 2F5 (ELDKWAS), but not the 4E10 epitope, were neutralization targets in two out of four viruses tested. Finally, the Fabs IgA from ESN women blocked efficiently transcytosis of HIV-1 with IC90 &lt;50-150 ng ml-1. However, all results showed good neutralizing activity of ESN Fabs. The efficiency of the Fabs in protecting the epithelial cells from free-HIV and CD4+ cells HIV-infectied, suggests that the low concentration of IgA, present in genital secretions, could be active in vivo. Our findings highlight important epitopes in gp41 that appear to be associated with exposure, but without infection, and would be important to consider for vaccine design. From these assumptions it has been possible to continue the study to design and produce immunogenic proteins which could elicit the humoral immune response, observed in the above categories. Thus we produced chimeric proteins, consisting of the carrier system of VLPs (Virus-Like Particles). In particular, have been used VLPs expressing regions and domains of HIV-gp41, produced in the first case in the baculovirus system, and in the second case, by the conjugation of gp41-peptides to VLPs, from bacteriophage Q?, AP205. All results showed that sera from immunized animals showed a high reactivity with individual HIV proteins expressed in VLPs. Results of TZM-bl based neutralization assay show that combined sera from animals independently immunized with gp140- or full-length-gp41-expressing VLPs have an additive/synergistic effect in the neutralization activity of HIV pseudoviruses. The peptides to which aminoacid strings were added to either the C-terminus or N-terminus of core epitope in HR1 region prior to VLP coupling could induced a strong specific immune response and HIV-1 neutralizing antibodies; high level of gp41 specific antibodies and neutralizing activity were also raised against MPR region. Antibody-dependent cell cytotoxicity (ADCC) activity was induced by one of the two MPR epitopes, whereas no ADCC activity was associated to HR1 region. These results may have relevant implications for the development of new vaccinal approaches against HIV infection.