This system is likely to be useful for a broader live attenuated YF 17D virus-based vaccine development for human diseases. Moreover, insertion of foreign genes into the flavivirus genome may also allow in vivo studies on flavivirus cell and tissue tropism as well as cellular processes related to flavivirus infection.

The yellow fever (YF) 17D vaccine is a live attenuated virus. Three-dimensional (3D) homology modeling of the E protein structure from YF 17D virus and its comparison with that from tick-borne encephalitis virus revealed that it is possible to accommodate inserts of different sizes and amino acid compositions in the flavivirus E protein fg loop. This is consistent with the 3D structures of both the dimeric and trimeric forms in which the fg loop lies exposed to solvents. We demonstrate here that YF 17D viruses bearing foreign humoral (17D/8) and T-cell (17D/13) epitopes, which vary in sequence and length, displayed growth restriction. It is hypothesized that interference with the dimer-trimer transition and with the formation of a ring of such trimers in order to allow fusion compromises the capability of the E protein to induce fusion of viral and endosomal membranes, and a slower rate of fusion may delay the extent of virus production. This would account for the lower levels of replication in cultured cells and of viremia in monkeys, as well as for the more attenuated phenotype of the recombinant viruses in monkeys. Testing of both recombinant viruses (17D/8 and 17D/13) for monkey neurovirulence also suggests that insertion at the 17D E protein fg loop does not compromise the attenuated phenotype of YF 17D virus, further confirming the potential use of this site for the development of new live attenuated 17D virus-based vaccines.The yellow fever (YF) 17D virus is attenuated and used for human vaccination. Some of the outstanding properties of this vaccine include limited viral replication in the host but with significant expansion and dissemination of the viral mass, yielding a robust and long-lived neutralizing antibody response. The vaccine is cheap and applied as a single dose, and there are well-established production methodology and quality control procedures, which include the monkey neurovirulence test (MNVT). Altogether, the 17D virus has become very attractive as an expression vector for the development of new live attenuated vaccines.The development of infectious-clone technology has allowed the genetic manipulation of the YF 17D virus genome toward the expression of foreign genes. Different technical approaches to constructing recombinant viruses based on the YF 17D virus are possible and will differ according to the antigen to be expressed. One major approach has been the creation of chimeric viruses through the exchange of structural prM/M/E genes (reviewed in reference 10). An alternative approach used in the development of YF 17D virus as a vector for heterologous antigens is the expression of particular epitopes in the E protein. In the mature virus, the E protein forms a symmetrical network of 90 dimers. These dimers are anchored into the viral envelope and lie flat on its surface. Each monomer is composed of three domains. The central and dimerization domains (I and II, respectively) are formed of several noncontiguous stretches of the polypeptide chain, while the C-terminal domain III is a c...

This work aims at the technological and innovative development of a noninjectable vaccine against a American Tegumentary Leishmaniasis (ATL), a neglected disease highly endemic in Brazil, leishmaniasis. Previous studies in the murine model demonstrated the efficacy of the intranasal route for the effective release of crude antigens (total lysate of Leishmania amazonensis-LaAg promastigotes) and DNA (LACK DNA) in the protection against cutaneous (L. (L.) amazonensis) and visceral (L. (L.) infantum) leishmaniasis. As most strains of mice are resistant to infection by species of the subgenus Viannia (L. braziliensis and L. guyanensis), the main responsible for cutaneous leishmaniasis in America (LTA), our group recently established the golden hamster L. braziliensis model for the study of pathogenesis and vaccine protection for ATL. We demonstrated the effectiveness of the intranasal vaccine with LaAg against infection by L. braziliensis hamster model. We have previously evidence that antigens called LVAL (not allowed disclosure-potential of patentability) could induce in vitro well modulated response in human cells. Then we hipotesize LVAL antigen could be a vaccine candidate against ATL. Objective: Thus, this work aims to evaluate the intranasal vaccine efficacy of the LVAL antigen and evaluate the immunological potential of the immunodominant fractions against infection by L. (V.) braziliensis in the hamster model. This study is part of a project with license number L7/17, approved by the CEUA/IOC-Fiocruz. Methodology: Hamsters were immunized with two doses of 20 μg LaAg or LVAL either intranasally or intramuscularly, with a 14-day interval between doses. The control group received PBS. After 14 days of the second immunization, the hamsters were infected on the dorsum of hind paw with 1 x 10 5 promastigotes of L. braziliensis. The lesion development was monitored weekly through the morphometry of the infected paw compared to the contralateral paw. Immunoblot methodology was performed to identify immunodominant antigenic fractions of the LaAg and LVAL antigens using serum samples from patients who evolved to cure spontaneous or posttreatment of LTA. Results: Hamsters vaccinated with LaAg and LVAL intramuscularly were not protected. The percentage of hamsters vaccinated with LaAg and LVAL that were considered protected (nodular lesions less than 1mm thick) was 50% and 43%, respectively, compared to 17% in the control group. Based on the immunoblot analysis, the fractions of the LVAL soluble antigens most frequently recognized by antibodies from sera from cured LTA individuals had molecular weight between 40 and 70 kDa. The molecules related to these bands will be fractionated and characterized biochemically. Conclusion: The identification and characterization of promising vaccine antigens may contribute to the definition of an active antigenic formulation in the protection against leishmaniasis that may serve for subsequent studies and evaluation of its potential clinical application.