Both T cells and B cells have been shown to be generated after infection with SARS-CoV-2 yet protocols or experimental models to study one or the other are less common. Here, we generate a chimeric protein (SpiN) that comprises the receptor binding domain (RBD) from Spike (S) and the nucleocapsid (N) antigens from SARS-CoV-2. Memory CD4+ and CD8+ T cells specific for SpiN could be detected in the blood of both individuals vaccinated with Coronavac SARS-CoV-2 vaccine and COVID-19 convalescent donors. In mice, SpiN elicited a strong IFN-γ response by T cells and high levels of antibodies to the inactivated virus, but not detectable neutralizing antibodies (nAbs). Importantly, immunization of Syrian hamsters and the human Angiotensin Convertase Enzyme-2-transgenic (K18-ACE-2) mice with Poly ICLC-adjuvanted SpiN promotes robust resistance to the wild type SARS-CoV-2, as indicated by viral load, lung inflammation, clinical outcome and reduction of lethality. The protection induced by SpiN was ablated by depletion of CD4+ and CD8+ T cells and not transferred by antibodies from vaccinated mice. Finally, vaccination with SpiN also protects the K18-ACE-2 mice against infection with Delta and Omicron SARS-CoV-2 isolates. Hence, vaccine formulations that elicit effector T cells specific for the N and RBD proteins may be used to improve COVID-19 vaccines and potentially circumvent the immune escape by variants of concern.
The current COVID-19 vaccines protect against severe disease, but are not effective in controlling replication of the Variants of Concern (VOCs). Here, we used the existing pre-clinical models of severe and moderate COVID-19 to evaluate the efficacy of a Spike-based DNA vaccine (pCTV-WS) for protection against different VOCs. Immunization of transgenic (K18-hACE2) mice and hamsters induced significant levels of neutralizing antibodies (nAbs) to Wuhan and Delta isolates, but not to the Gamma and Omicron variants. Nevertheless, the pCTV-WS vaccine offered significant protection to all VOCs. Consistently, protection against lung pathology and viral load to Wuhan or Delta was mediated by nAbs, whereas in the absence of nAbs, T cells controlled viral replication, disease and lethality in mice infected with either the Gamma or Omicron variants. Hence, considering the conserved nature of CD4 and CD8 T cell epitopes, we corroborate the hypothesis that induction of effector T-cells should be a main goal for new vaccines against the emergent SARS-CoV-2 VOCs.
The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFNγ response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies. Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. Thus, the N protein should be considered to induce T-cell-based immunity to improve SARS-CoV-2 vaccines, and eventually to circumvent the immune scape by variants.
The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFNγ response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies (nAb). Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. This protective immunity was dependent of CD4+ T and CD8+ T cells, but not by transfer of antibody of vaccinated mice. Thus, our experiment provides an example T cell-mediated immunity and reinforce the concept that T cell target antigens other that the S protein maybe considered to improve SARS-CoV-2 vaccines, and eventually circumvent the immune scape by variants.
ResumoA Síndrome Neuroléptica Maligna é uma rara complicação provocada pelos agentes neurolépticos, relacionandose ao bloqueio dos receptores dopaminérgicos nos gânglios da base. É conhecida como síndrome da deficiência aguda da dopamina e pode ser caracterizada por hiperpirexia, alteração do nível de consciência, hipertonia, disfunção autonômica, insuficiência respiratória, rabdomiólise e leucocitose. MDFG, cinquenta anos, sexo feminino, deu entrada em um hospital psiquiátrico apresentando ideias persecutórias, seguida de perda de consciência associada a quadro de convulsão tônico-clônica, sendo internada por quinze dias sob medicação específica. Aos dez dias de internação, apresentou rigidez muscular associada à hipertermia, disartria, disfagia, sialorreia, ataxia, dispneia e confusão mental. Foram realizados exames bioquímicos, hematológicos, microbiológicos e imagiológicos; apenas a enzima Gama Glutamil Transferase (GGT) apresentou-se aumentada. A paciente foi liberada sem melhora do quadro secundário e o mesmo regime terapêutico foi administrado em domicílio. Três dias após a alta hospitalar, devido ao agravamento sintomatológico, a paciente procurou pela emergência e novos exames laboratoriais foram realizados. Nesse episódio outros exames apresentaram-se alteradas: GGT, Creatino Fosfoquinase (CPK), Bilirrubina Direta e Urobilinogênio aumentados. O regime terapêutico foi alterado; dois meses após a suspensão da medicação inicial, outros exames foram realizados sem apresentar alteração. Sabe-se que determinados fármacos induzem a síntese e aumento da atividade plasmática da GGT, bem como o aumento da síntese de bilirrubina direta e excreção de urobilinogênio na urina. Além disso, o aumento da CK está associado à lesão do músculo esquelético, característica na SNM, uma vez que a atividade terapêutica dos antipsicóticos está relacionada ao bloqueio da dopamina nos receptores do tipo D2 no tegmento ventral e substância negra. Essa é a principal explicação da fisiopatogenia da doença. Assim, os achados laboratoriais associados à sintomatologia da paciente e à reversão do quadro após suspensão da medicação, sugerem quadro de SNM. Palavras-chave: Antipsicóticos. Dopamina. Gânglios da base.Artigo recebido em 08 de Junho de 2016 e aprovado em 30 de Agosto de 2017.
Introduction:The disease called COVID-19 emerged in China in December 2019 and was recognized by the World Health Organization (WHO) as a pandemic in March 2020. Given the high number of cases and deaths worldwide, the emergence of new variants may require specific vaccines for each country/region. Therefore, countries with technology parks could become self-sufficient to produce its own vaccines.Objective: The present study aimed to develop a Brazilian DNA-based vaccine for COVID-19 using recombinant plasmids carrying the gene sequence of the Spike protein from SARS-CoV-2.Methodology: Spike gene sequence was cloned into the pCTV (expression plasmid), transformed into DH5α bacteria and purified using Plasmid Giga Kit (Qiagen). Mice were immunized with two intramuscular doses (21 days apart) containing 100 ug of DNA. The specific humoral response was evaluated by total IgG, IgG subclasses (IgG1 and IgG2c) and neutralizing antibody titers in plasma samples. Splenocytes were stimulated with Wuhan strain recombinant protein (RBD domain) for detection of IFN-γ. For protection evaluation, hACE-transgenic mice were immunized and challenged with SARS-CoV-2.Results: High levels of total IgG and IgG2c were detected in immunized animals using different plasma dilutions (1:25 until 1:25,000; (p≤0.0001). Regarding cellular immune response, animals immunized with pCTV Spike showed high IFN-γ secretion in response to specific stimulation with RBD (p<0.05) compared to control groups. Immunized animals showed high percentage of neutralization (nAb). All immunized animals survived after challenge with SARS-CoV-2 and no viral load was detected in the lung tissue after 5 days of the challenge (measured by PFU) (p<0.001). The histopathological analyses reveal the presence of edema, vascular congestion, hemorrhage and intense inflammatory infiltrate in the lungs of non-immunized animals. Conclusion:These findings suggest that the pCTV Spike could be an interesting vaccine against SARS-CoV-2, since it induces a strong immune response and protect all challenged animals. The next step is to evaluate the protection rate induced by DNA containing the Spike gene sequence in immunized hACEtransgenic mice and challenged with SARS-CoV-2 variants.
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