Phenotypic and Genetic Studies of the Viral Lineage Associated with the Recent Yellow Fever Outbreak in Brazil

Yellow fever virus (YFV) caused an outbreak in the Brazilian Southeast from 2016 to 2019, of the most significant magnitude since the 1900s. An investigation of the circulating virus revealed that most of the genomes detected in this period carried nine unique amino acid polymorphisms, with eight located in the non-structural proteins NS3 and NS5, which are pivotal for viral replication. To elucidate the effect of these amino acid changes on viral infection, we constructed viruses carrying amino acid alterations in NS3 and NS5, performed infection in different cells, and assessed their neurovirulence in BALB/c mice and infected AG129 mice. We observed that the residues that compose the YFV 2016–2019 molecular signature in the NS5 protein might have been related to an attenuated phenotype, and that the alterations in the NS3 protein only slightly affected viral infection in AG129 mice, increasing to a low extent the mortality rate of these animals. These results contributed to unveiling the role of specific naturally occurring amino acid changes in the circulating strain of YFV in Brazil

Retention of a recombinant GFP protein expressed by the yellow fever 17D virus in the E/NS1 intergenic region in the endoplasmic reticulum

The flaviviral envelope proteins, E protein and precursor membrane protein, are mainly associated with the endoplasmic reticulum (ER) through two transmembrane (TM) domains that are exposed to the luminal face of this compartment. Their retention is associated with the viral assembly process. ER-retrieval motifs were mapped at the carboxy terminus of these envelope proteins. A recombinant yellow fever (YF) 17D virus expressing the reporter green fluorescent protein (GFP) with the stem-anchor (SA) region of E protein fused to its carboxy terminus was subjected to distinct genetic mutations in the SA sequence to investigate their effect on ER retention. Initially, we introduced progressive deletions of the stem elements (H1, CS and H2). In a second set of mutants, the effect of a length increase for the first TM anchor region was evaluated either by replacing it with the longer TM of human LAMP-1 or by the insertion of the VALLLVA sequence into its carboxy terminus. We did not detect any effect on the GFP localisation in the cell, which remained associated with the ER. Further studies should be undertaken to elucidate the causes of the ER retention of recombinant proteins expressed at the intergenic E/NS1 region of the YF 17D virus polyprotein

Recombinant Yellow Fever Viruses Elicit CD8(+) T Cell Responses and Protective Immunity against Trypanosoma cruzi

Chagas' disease is a major public health problem affecting nearly 10 million in Latin America. Despite several experimental vaccines have shown to be immunogenic and protective in mouse models, there is not a current vaccine being licensed for humans or in clinical trial against T. cruzi infection. Towards this goal, we used the backbone of Yellow Fever (YF) 17D virus, one of the most effective and well-established human vaccines, to express an immunogenic fragment derived from T. cruzi Amastigote Surface Protein 2 (ASP-2). the cDNA sequence of an ASP-2 fragment was inserted between E and NS1 genes of YF 17D virus through the construction of a recombinant heterologous cassette. the replication ability and genetic stability of recombinant YF virus (YF17D/ENS1/Tc) was confirmed for at least six passages in Vero cells. Immunogenicity studies showed that YF17D/ENS1/Tc virus elicited neutralizing antibodies and gamma interferon (IFN-gamma) producing-cells against the YF virus. Also, it was able to prime a CD8(+) T cell directed against the transgenic T. cruzi epitope (TEWETGQI) which expanded significantly as measured by T cell-specific production of IFN-gamma before and after T. cruzi challenge. However, most important for the purposes of vaccine development was the fact that a more efficient protective response could be seen in mice challenged after vaccination with the YF viral formulation consisting of YF17D/ENS1/Tc and a YF17D recombinant virus expressing the TEWETGQI epitope at the NS2B-3 junction. the superior protective immunity observed might be due to an earlier priming of epitope-specific IFN-gamma-producing T CD8(+) cells induced by vaccination with this viral formulation. Our results suggest that the use of viral formulations consisting of a mixture of recombinant YF 17D viruses may be a promising strategy to elicit protective immune responses against pathogens, in general

Induction of IFN-γ secreting splenocytes in vaccinated mice before and after <i>T. cruzi</i> challenge.

<p>Groups of A/J mice were immunized once or twice with medium (mock) (○), YF 17DD vaccine (□), YF17D/NS2B3/Tc virus (•) and YF17D/ENS1/Tc virus (▾) or alternatively with Formulation 1 (50,000 PFU of YF17D/NS2B3/Tc and 50,000 PFU of YF17D/ENS1/Tc) (♦) and challenged or not with 250 <i>T. cruzi</i> trypomastigotes. Spleen cells of each mouse were obtained one week after the first dose (A and C), one week after the second dose (B and D) or two weeks after challenge (E) and after that, were stimulated <i>in vitro</i> with YF 17DD inactivated virus (A and B) or TEWETGQI-peptide (C, D and E) to assess cellular responses to YF peptides or to the <i>T. cruzi</i> peptide, respectively. Results represent IFN-γ producing cells (SFC) per 10⁶ spleen cells. Asterisks indicate statistically significant differences between groups of immunization in comparison to Mock (A and B) or in comparison to Mock and YF 17DD group (C, D and E) and were done by ANOVA Tukeýs test (***<i>P</i><0.0001; **<i>P</i><0.01; * <i>P</i><0.05).</p

Expression of the <i>T. cruzi</i> TEWETGQI epitope by the recombinant YF17D/ENS1/Tc virus.

<p>Indirect immunofluorescence assay of Vero cells infected with YF 17DD vaccine virus (A and D), recombinant YF17D/ENS1/Tc viral (B and E), and recombinant YF17D/NS2B3/Tc virus (C and F). Cells were stained with a mouse polyclonal hyperimmune serum to YF 17D (panels A, B and C) or a polyclonal antibodies directed to the TEWETGQI epitope (panels D, E and F). The employed secondary antibodies were labeled with Alexa Fluor 488 (A, B and C) and Alexa Fluor 546 (D, E and F).</p

Immunogenicity of YF 17D recombinant viruses or viral formulations in A/J mice.

a<p>50% of YF17D/ENS1/Tc and 50% of YF17D/NS2B3/Tc viruses.</p>b<p>75% of YF17D/ENS1/Tc and 25% of YF17D/NS2B3/Tc viruses.</p>*<p>values indicate the reciprocal of the dilution yielding 50%. PRNT, Plaque Reduction Neutralization Test; GMT, Geometric Mean Titre.</p>**<p>differences in the titers of neutralizing antibodies virus between YF 17DD and the recombinant viruses or viral formulations were statistically significant (<i>P</i> value <0.05; Kruskal-Wallis test).</p