Biological and immunological characterization of recombinant Yellow Fever 17D Viruses expressing a Trypanosoma cruzi Amastigote Surface Protein-2 CD8+ T cell epitope at two distinct regions of the genome

<p>Abstract</p> <p>Background</p> <p>The attenuated Yellow fever (YF) 17D vaccine virus is one of the safest and most effective viral vaccines administered to humans, in which it elicits a polyvalent immune response. Herein, we used the YF 17D backbone to express a <it>Trypanosoma cruzi </it>CD8⁺T cell epitope from the Amastigote Surface Protein 2 (ASP-2) to provide further evidence for the potential of this virus to express foreign epitopes. The TEWETGQI CD8⁺T cell epitope was cloned and expressed based on two different genomic insertion sites: in the <it>fg </it>loop of the viral Envelope protein and the protease cleavage site between the NS2B and NS3. We investigated whether the site of expression had any influence on immunogenicity of this model epitope.</p> <p>Results</p> <p>Recombinant viruses replicated similarly to vaccine virus YF 17D in cell culture and remained genetically stable after several serial passages in Vero cells. Immunogenicity studies revealed that both recombinant viruses elicited neutralizing antibodies to the YF virus as well as generated an antigen-specific gamma interferon mediated T-cell response in immunized mice. The recombinant viruses displayed a more attenuated phenotype than the YF 17DD vaccine counterpart in mice. Vaccination of a mouse lineage highly susceptible to infection by <it>T. cruzi </it>with a homologous prime-boost regimen of recombinant YF viruses elicited TEWETGQI specific CD8⁺T cells which might be correlated with a delay in mouse mortality after a challenge with a lethal dose of <it>T. cruzi</it>.</p> <p>Conclusions</p> <p>We conclude that the YF 17D platform is useful to express <it>T. cruzi </it>(Protozoan) antigens at different functional regions of its genome with minimal reduction of vector fitness. In addition, the model <it>T. cruzi </it>epitope expressed at different regions of the YF 17D genome elicited a similar T cell-based immune response, suggesting that both expression sites are useful. However, the epitope as such is not protective and it remains to be seen whether expression of larger domains of ASP-2, which include the TEWETGQI epitope, will elicit better T-CD8+ responses to the latter. It is likely that additional antigens and recombinant virus formulations will be necessary to generate a protective response.</p

Fetal Demise and Failed Antibody Therapy During Zika Virus Infection of Pregnant Macaques

Zika virus (ZIKV) infection of pregnant women is associated with pathologic complications of fetal development. Here, we infect pregnant rhesus macaques (Macaca mulatta) with a minimally passaged ZIKV isolate from Rio de Janeiro, where a high rate of fetal development complications was observed. The infection of pregnant macaques with this virus results in maternal viremia, virus crossing into the amniotic fluid (AF), and in utero fetal deaths. We also treated three additional ZIKV-infected pregnant macaques with a cocktail of ZIKV-neutralizing human monoclonal antibodies (nmAbs) at peak viremia. While the nmAbs can be effective in clearing the virus from the maternal sera of treated monkeys, it is not sufficient to clear ZIKV from AF. Our report suggests that ZIKV from Brazil causes fetal demise in non-human primates (NHPs) without additional mutations or confounding co-factors. Treatment with a neutralizing anti-ZIKV nmAb cocktail is insufficient to fully stop vertical transmission

DNA Vaccines against Dengue Virus Type 2 Based on Truncate Envelope Protein or Its Domain III

Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2

GagCM9-Specific CD8+ T Cells Expressing Limited Public TCR Clonotypes Do Not Suppress SIV Replication In Vivo

Several lines of evidence suggest that HIV/SIV-specific CD8+ T cells play a critical role in the control of viral replication. Recently we observed high levels of viremia in Indian rhesus macaques vaccinated with a segment of SIVmac239 Gag (Gag45–269) that were subsequently infected with SIVsmE660. These seven Mamu-A*01+ animals developed CD8+ T cell responses against an immunodominant epitope in Gag, GagCM9, yet failed to control virus replication. We carried out a series of immunological and virological assays to understand why these Gag-specific CD8+ T cells could not control virus replication in vivo. GagCM9-specific CD8+ T cells from all of the animals were multifunctional and were found in the colonic mucosa. Additionally, GagCM9-specific CD8+ T cells accessed B cell follicles, the primary residence of SIV-infected cells in lymph nodes, with effector to target ratios between 20–250 GagCM9-specific CD8+ T cells per SIV-producing cell. Interestingly, vaccinated animals had few public TCR clonotypes within the GagCM9-specific CD8+ T cell population pre- and post-infection. The number of public TCR clonotypes expressed by GagCM9-specific CD8+ T cells post-infection significantly inversely correlated with chronic phase viral load. It is possible that these seven animals failed to control viral replication because of the narrow TCR repertoire expressed by the GagCM9-specific CD8+ T cell population elicited by vaccination and infection

Rare Control of SIVmac239 Infection in a Vaccinated Rhesus Macaque.

Effector memory T cell (TEM) responses display potent antiviral properties and have been linked to stringent control of simian immunodeficiency virus (SIV) replication. Since recurrent antigen stimulation drives the differentiation of CD8+ T cells toward the TEM phenotype, in this study we incorporated a persistent herpesviral vector into a heterologous prime/boost/boost vaccine approach to maximize the induction of TEM responses. This new regimen resulted in CD8+ TEM-biased responses in four rhesus macaques, three of which controlled viral replication to <1,000 viral RNA copies/ml of plasma for more than 6 months after infection with SIVmac239. Over the course of this study, we made a series of interesting observations in one of these successful controller animals. Indeed, in vivo elimination of CD8αβ+ T cells using a new CD8β-depleting antibody did not abrogate virologic control in this monkey. Only after its CD8α+ lymphocytes were depleted did SIV rebound, suggesting that CD8αα+ but not CD8αβ+ cells were controlling viral replication. By 2 weeks postinfection (PI), the only SIV sequences that could be detected in this animal harbored a small in-frame deletion in nef affecting six amino acids. Deep sequencing of the SIVmac239 challenge stock revealed no evidence of this polymorphism. However, sequencing of the rebound virus following CD8α depletion at week 38.4 PI again revealed only the six-amino acid deletion in nef. While any role for immunological pressure on the selection of this deleted variant remains uncertain, our data provide anecdotal evidence that control of SIV replication can be maintained without an intact CD8αβ+ T cell compartment

Post-acute COVID-19 syndrome after reinfection and vaccine breakthrough by the SARS-CoV-2 Gamma variant in Brazil.

We describe a case of prolonged COVID-19 caused by the SARS-CoV-2 Gamma variant in a fully vaccinated healthcare worker, 387 days after an infection caused by lineage B.1.1.33. Infections were confirmed by whole-genome sequencing and corroborated by the detection of neutralizing antibodies in convalescent serum samples. Considering the permanent exposure of this healthcare worker to SARS-CoV-2, the waning immunity after the first infection, the low efficacy of the inactivated vaccine at preventing COVID-19, the immune escape of the Gamma variant (VOC), and the burden of post-COVID syndrome, this individual would have benefited from an additional dose of a heterologous vaccine

Repertoire, Genealogy and Genomic Organization of Cruzipain and Homologous Genes in Trypanosoma cruzi, T. cruzi-Like and Other Trypanosome Species

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches

Study protocol for the multicentre cohorts of Zika virus infection in pregnant women, infants, and acute clinical cases in Latin America and the Caribbean: The ZIKAlliance consortium

Background: The European Commission (EC) Horizon 2020 (H2020)-funded ZIKAlliance Consortium designed a multicentre study including pregnant women (PW), children (CH) and natural history (NH) cohorts. Clinical sites were selected over a wide geographic range within Latin America and the Caribbean, taking into account the dynamic course of the ZIKV epidemic. Methods: Recruitment to the PW cohort will take place in antenatal care clinics. PW will be enrolled regardless of symptoms and followed over the course of pregnancy, approximately every 4 weeks. PW will be revisited at delivery (or after miscarriage/abortion) to assess birth outcomes, including microcephaly and other congenital abnormalities according to the evolving definition of congenital Zika syndrome (CZS). After birth, children will be followed for 2 years in the CH cohort. Follow-up visits are scheduled at ages 1-3, 4-6, 12, and 24 months to assess neurocognitive and developmental milestones. In addition, a NH cohort for the characterization of symptomatic rash/fever illness was designed, including follow-up to capture persisting health problems. Blood, urine, and other biological materials will be collected, and tested for ZIKV and other relevant arboviral diseases (dengue, chikungunya, yellow fever) using RT-PCR or serological methods. A virtual, decentralized biobank will be created. Reciprocal clinical monitoring has been established between partner sites. Substudies of ZIKV seroprevalence, transmissio