Immunogenicity and reactogenicity of yellow fever vaccine in people with HIV.

OBJECTIVE: To evaluate immunogenicity and reactogenicity of yellow fever (YF) vaccine in people with HIV (PWH) compared to HIV-uninfected controls. DESIGN: In this longitudinal interventional trial (NCT03132311), PWH with CD4 + cell count ≥200 cells/µl and controls, aged 18-59, without a previous history of YF vaccination received a single standard dose of YF vaccine (17DD) and were followed at Days 5, 30 and Year 1. METHODS: YF-neutralization titers were measured at Days 0, 30 and Year 1 and geometric mean titers (GMT) were calculated. Adverse events (AE) and YF virus detection were measured at Days 5 and 30. Linear regression evaluated factors associated with YF-neutralization titers. RESULTS: Two hundred and eighteen PWH and 82 controls were included. At baseline, all PWH were using antiretroviral therapy; 92.6% had undetectable HIV viral load (VL) and median CD4 + cell count was 630 cells/µl [interquartile range (IQR) 463-888]. YF vaccine was safe and there were no serious AEs. At Day 30, seroconversion was observed in 98.6% of PWH [95% confidence interval (CI): 95.6-99.6] and in 100% of controls (95% CI: 93.9-100); at Year 1, 94.0% of PWH (95% CI: 89.6-96.7) and 98.4% of controls (95% CI 90.3-99.9) were seropositive. PWH had lower GMTs than controls at Day 30 and Year 1. Baseline VL >1000 copies/ml, low CD4 + cell count and low CD4 + /CD8 + ratio were associated with lower YF-neutralization titers. CONCLUSIONS: YF vaccine is safe in PWH with CD4 + cell count ≥200 cells/µl. YF vaccine immunogenicity is impaired in PWH, particularly among those with high VL, low CD4 + cell count and low CD4 + /CD8 + ratio at vaccination and YF-neutralization titers decays over time.

Two-Step In Vitro Model to Evaluate the Cellular Immune Response to SARS-CoV-2.

The cellular immune response plays an important role in COVID-19, caused by SARS-CoV-2. This feature makes use of in vitro models' useful tools to evaluate vaccines and biopharmaceutical effects. Here, we developed a two-step model to evaluate the cellular immune response after SARS-CoV-2 infection-induced or spike protein stimulation in peripheral blood mononuclear cells (PBMC) from both unexposed and COVID-19 (primo-infected) individuals (Step1). Moreover, the supernatants of these cultures were used to evaluate its effects on lung cell lines (A549) (Step2). When PBMC from the unexposed were infected by SARS-CoV-2, cytotoxic natural killer and nonclassical monocytes expressing inflammatory cytokines genes were raised. The supernatant of these cells can induce apoptosis of A549 cells (mock vs. Step2 [mean]: 6.4% × 17.7%). Meanwhile, PBMCs from primo-infected presented their memory CD4+ T cells activated with a high production of IFNG and antiviral genes. Supernatant from past COVID-19 subjects contributed to reduce apoptosis (mock vs. Step2 [ratio]: 7.2 × 1.4) and to elevate the antiviral activity (iNOS) of A549 cells (mock vs. Step2 [mean]: 31.5% × 55.7%). Our findings showed features of immune primary cells and lung cell lines response after SARS-CoV-2 or spike protein stimulation that can be used as an in vitro model to study the immunity effects after SARS-CoV-2 antigen exposure.

Sofosbuvir shows a protective effect against vertical transmission of Zika virus and the associated congenital syndrome in rhesus monkeys.

The outbreaks of Zika virus (ZIKV) infection in Brazil, 2015-2016, were associated with severe congenital malformations. Our translational study aimed to test the efficacy of the antiviral agent sofosbuvir (SOF) against vertical transmission of ZIKV and the associated congenital syndrome (CZS), using a rhesus monkey model. Eight pregnant macaques were successfully infected during the organogenesis phase with a Brazilian ZIKV strain; five of them received SOF from two to fifteen days post-infection. Both groups of dams showed ZIKV-associated clinical signals, detectable ZIKV RNA in several specimens, specific anti-ZIKV IgM and IgG antibodies, and maternal neutralizing antibodies. However, malformations occurred only among non-treated dam offspring. Compared to non-treated animals, all SOF-treated dams had a shorter ZIKV viremia and four of five neonates had undetectable ZIKV RNA in blood and tissue samples. These results support further clinical evaluations aiming for the prevention of CZS.

Dissecting the role of protein-protein and protein-nucleic acid interactions in MS2 bacteriophage stability.

To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.

Mutations in the hydrophobic core and in the protein-RNA interface affect the packing and stability of icosahedral viruses.

The information required for successful assembly of an icosahedral virus is encoded in the native conformation of the capsid protein and in its interaction with the nucleic acid. Here we investigated how the packing and stability of virus capsids are sensitive to single amino acid substitutions in the coat protein. Tryptophan fluorescence, bis-8-anilinonaphthalene-1-sulfonate fluorescence, CD and light scattering were employed to measure urea- and pressure-induced effects on MS2 bacteriophage and temperature sensitive mutants. M88V and T45S particles were less stable than the wild-type forms and completely dissociated at 3.0 kbar of pressure. M88V and T45S mutants also had lower stability in the presence of urea. We propose that the lower stability of M88V particles is related to an increase in the cavity of the hydrophobic core. Bis-8-anilinonaphthalene-1-sulfonate fluorescence increased for the pressure-dissociated mutants but not for the urea-denatured samples, indicating that the final products were different. To verify reassembly of the particles, gel filtration chromatography and infectivity assays were performed. The phage titer was reduced dramatically when particles were treated with a high concentration of urea. In contrast, the phage titer recovered after high-pressure treatment. Thus, after pressure-induced dissociation of the virus, information for correct reassembly was preserved. In contrast to M88V and T45S, the D11N mutant virus particle was more stable than the wild-type virus, in spite of it also possessing a temperature sensitive growth phenotype. Overall, our data show how point substitutions in the capsid protein, which affect either the packing or the interaction at the protein-RNA interface, result in changes in virus stability.