Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load. METHODS: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen. RESULTS: T CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42-50 and HBZ157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA. CONCLUSIONS: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.

The Perennial Use of the Green Fluorescent Protein Marker in a Live Vaccinia Virus Ankara Recombinant Platform Shows No Acute Adverse Effects in Mice.

Recombinant virus vectors represent a promising strategy for vaccine research. Among available viral vectors, members of the Poxviridae family-especially the modified Vaccinia virus Ankara (MVA)-stand out as immunogenic and safe vaccine platforms. Because MVA usually does not produce plaques in cell culture, visible selection markers such as the green fluorescent protein (GFP) are frequently incorporated into the constructions in order to facilitate the recognition of recombinants. However, these genetic markers have to be removed before any clinical trial. Here, we evaluated the acute responses generated in mice immunized with a MVA vector in which the GFP marker was not removed. We observed no differences in neutrophil, monocyte, or total leucocyte recruitment among animals inoculated with MVA or MVA-GFP. Likewise, there were no differences in neutrophil activation between mice groups. Hepatic functions were not altered in either MVA or MVA-GFP-inoculated mice, and we observed no histopathological alterations in different tissues from virus-inoculated animals. In conclusion, the presence of GFP is innocuous to immunized animals and do not alter acute physiopathological responses to the MVA vector. We suggest that keeping the GFP marker may be a good strategy for vaccine development, production, and evaluation.