Evaluation of peptide based vaccines and inhibitors to prevent the onset of HTLV-1 associated diseases

by Lynch, Marcus Phillip.

The human T-cell lymphotropic virus (HTLV-1) is the etiologic agent of an aggressive T-cell malignancy, adult T-cell leukemia (ATL). HTLV-1 infected individuals can also develop a chronic inflammatory condition affecting the central nervous system termed HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), as well as other inflammatory disorders. Although only ? 10% of infected individuals develop some type of disease state and the majority of infected individuals remain asymptomatic, the underlying causes for disease predilection and predictive markers for disease progression are poorly understood. In addition there is a lack of effective treatment against the serious ailments associated with HTLV-1 infection. The purpose of this dissertation is to show three potential peptide-based modes of treatment to combat HTLV-1 infection. First prophylactic vaccination was investigated by using the squirrel monkey model of HTLV-1 to evaluate the protective efficacy of two B-cell epitope vaccine candidates MVF175-218 and WCCR2T. The chimeric MVF175-218 peptide containing a promiscuous T-cell epitope derived from the measles virus (MVF) derived from gp46; induced high titered antibody responses which bound to Env on the surface of HTLV-1 infected cells. WCCR2T, derived from the coiled-coil region, residues 347-374, of gp21 induced high titered antibodies in 1 of 4 immunized animals and 2 of 4 were shown to have antibodies that bound Env on the surface of HTLV-1 infected MT-2 cells. ii Monkeys were challenged with the EVO/1540 cell line; however all animals were able to lower the proviral load to undetectable levels in all but one animal by 31 weeks after challenge; therefore, the protective effects of the vaccines were unable to be evaluated. The second methodology investigated for use against HTLV-1 associated diseases was the identification and improvement of possible MHC I epitopes that could be used for CD8+ therapeutic vaccination. Predictive computer algorithms were used to identify the highest ranking HLA-A* 0201 restricted epitopes from the HTLV-1 Gag Pol, and Tax proteins. High ranking epitopes with sub-optimal anchor residues were evaluated along with mutant peptides of the epitope containing optimal anchor residues. Relative peptide affinity was measured using the T2 binding assay and immunogenicity was measured in HLA- A*0201 transgenic HHD II mice. In vitro cytotoxicity assays with effector CTL from peptide immunized mice were combined with HTLV-1 infected HLA-A*0201 positive Hut102 A2- GFP cells to determine which epitopes were expressed on the surface of HTLV-1 infected cells enough to sensitize the cells to lysis. Results from this assay and ELISPOT with PBMC from a HAM/TSP patient show that even though enhancement improves the immunogenicity of some of the peptides; the only epitope examined that was expressed enough on the surface to sensitize the cells to lysis was the well defined Tax 11-19 epitope. These findings have important implications for further studies that may want to pursue CD8+ immunotherapy to control HTLV-1 infection. The final mode investigated was peptide fusion inhibitors. To improve the half-life of potential HTLV-1 fusion inhibitors a retro-inverso peptide mimetic RI P400 was made was synthesized and compared to the previously described native P400 peptide. Circular dichroism measurements showed that the peptides had very similar mirror image secondary iii structure and % helicity. P400 and RI P400 both showed strong syncytia inhibition at 100 µM peptide concentration. However the efficacy of RI P400 declined quickly with decreasing concentration. A longer peptide encompassing the P197 region was made covering residues 175-218 to see if extra length would increase syncytia inhibition in the gp46 peptide. However it was shown that P197 was sufficient to inhibit syncytia, and the larger peptide had slightly lower inhibitory capacity than P197. Next P197 and P400 were combined 1:1 in and compared with equal molar concentrations the individual peptides. It was shown that there was no synergism, or additive effect, when the peptides were combined. Finally rational mutants in the 401 position of P400 were evaluated. Shortened peptides 22 aa in length were synthesized on a multiple peptide synthesizer. These peptides were better than the negative control but lost their inhibitory capacity. Therefore, rational mutant inhibitors should be evaluated further as full length 30 aa peptide inhibitors, as smaller peptides were ineffective. This dissertation shows three different peptide-based approaches that could be used to combat HTLV-1 infection and associated diseases including prophylactic as well as therapeutic approaches. iv