RESUMO
During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.
Assuntos
COVID-19 , Saccharomycetales , Vacinas , Humanos , COVID-19/diagnóstico , COVID-19/prevenção & controle , Teste para COVID-19 , Pichia/genética , Pichia/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Proteínas Recombinantes/química , Vacinas/metabolismo , Anticorpos Neutralizantes/metabolismo , Anticorpos AntiviraisRESUMO
SARS-CoV-2 variant Lambda was dominant in several South American countries, including Chile. To ascertain the efficacy of local vaccination efforts, we used pseudotyped viruses to characterize the neutralization capacity of antibodies elicited by CoronaVac (n = 53) and BNT162b2 (n = 56) in healthcare workers from Clínica Santa María and the Faculty of Medicine at Universidad de Chile, as well as in convalescent plasma from individuals infected during the first wave visiting the Hospital Clínico at Pontificia Universidad Católica (n = 30). We observed that BNT162b2 elicits higher neutralizing antibody titres than CoronaVac, with differences ranging from 7.4-fold for the ancestral spike (Wuhan-Hu-1) to 8.2-fold for the Lambda spike and 13-fold for the Delta spike. Compared with the ancestral virus, neutralization against D614G, Alpha, Gamma, Lambda and Delta variants was reduced by between 0.93- and 4.22-fold for CoronaVac, 1.04- and 2.38-fold for BNT162b2, and 1.26- and 2.67-fold for convalescent plasma. Comparative analyses among the spike structures of the different variants suggest that mutations in the spike protein from the Lambda variant, including the 246-252 deletion in an antigenic supersite at the N-terminal domain loop and L452Q/F490S within the receptor-binding domain, may account for immune escape. Interestingly, analyses using pseudotyped and whole viruses showed increased entry rates into HEK293T-ACE2 cells, but reduced replication rates in Vero-E6 cells for the Lambda variant when compared with the Alpha, Gamma and Delta variants. Our data show that inactivated virus and messenger RNA vaccines elicit different levels of neutralizing antibodies with different potency to neutralize SARS-CoV-2 variants, including the variant of interest Lambda.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes/metabolismo , Vacina BNT162 , COVID-19/terapia , Chile , Células HEK293 , Humanos , Imunização Passiva , Glicoproteínas de Membrana/metabolismo , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas do Envelope Viral/metabolismo , Soroterapia para COVID-19RESUMO
For over a century, polyclonal antibodies have been used to treat snakebite envenoming and are still considered by the WHO as the only scientifically validated treatment for snakebites. Nevertheless, moderate innovations have been introduced to this immunotherapy. New strategies and approaches to understanding how antibodies recognize and neutralize snake toxins represent a challenge for next-generation antivenoms. The neurotoxic activity of Micrurus venom is mainly due to two distinct protein families, three-finger toxins (3FTx) and phospholipases A2 (PLA2). Structural conservation among protein family members may represent an opportunity to generate neutralizing monoclonal antibodies (mAbs) against family-conserved epitopes. In this work, we sought to produce a set of monoclonal antibodies against the most toxic components of M. altirostris venom. To this end, the crude venom was fractionated, and its major toxic proteins were identified and used to generate a panel of five mAbs. The specificity of these mAbs was characterized by ELISA and antivenomics approaches. Two of the generated mAbs recognized PLA2 epitopes. They inhibited PLA2 catalytic activity and showed paraspecific neutralization against the myotoxicity from the lethal effect of Micrurus and Naja venoms' PLA2s. Epitope conservation among venom PLA2 molecules suggests the possibility of generating pan-PLA2 neutralizing antibodies.
Assuntos
Cobras Corais , Mordeduras de Serpentes , Animais , Cobras Corais/metabolismo , Elapidae/metabolismo , Epitopos , Venenos Elapídicos/toxicidade , Antivenenos , Fosfolipases A2/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Monoclonais/metabolismoRESUMO
BACKGROUND: Interleukin (IL)-15 is a proinflammatory T-cell growth factor overexpressed in several autoimmune diseases such as rheumatoid arthritis. Our initial strategy to neutralize the increased levels of IL-15 consisted in a vaccine candidate based on the recombinant modified human IL-15 (mhIL-15) mixed with the alum adjuvant. A previous study in non-human primates Macaca fascicularis has shown that vaccination induces neutralizing antibodies against native IL-15, without affecting animal behavior, clinical status, or the percentage of IL-15-dependent cell populations. However, the mhIL-15 used as an antigen was active in the IL-2-dependent cytotoxic T-cell line CTLL-2, which could hinder its therapeutic application. The current article evaluated the immunogenicity in African green monkeys of a vaccine candidate based on IL-15 mutant D8SQ108S, an inactive form of human IL-15. RESULTS: IL-15 D8SQ108S was inactive in the CTLL-2 bioassay but was able to competitively inhibit the biological activity of human IL-15. Immunization with 200 µg of IL-15 mutant combined with alum elicited anti-IL-15 IgG antibodies after the second and third immunizations. The median values of anti-IL-15 antibody titers were slightly higher than those generated in animals immunized with 200 µg of mhIL-15. The highest antibody titers were induced after the third immunization in monkeys vaccinated with 350 µg of IL-15 D8SQ108S. In addition, sera from immunized animals inhibited the biological activity of human IL-15 in CTLL-2 cells. The maximum neutralizing effect was observed after the third immunization in sera of monkeys vaccinated with the highest dose of the IL-15 mutant. These sera also inhibited the proliferative activity of simian IL-15 in the CTLL-2 bioassay and did not affect the IL-2-induced proliferation of the aforementioned T-cell line. Finally, it was observed that vaccination neither affects the animal behavior nor the general clinical parameters of immunized monkeys. CONCLUSION: Immunization with inactive IL-15 D8SQ108S mixed with alum generated neutralizing antibodies specific for human IL-15 in African green monkeys. Based on this fact, the current vaccine candidate could be more effective than the one based on biologically active mhIL-15 for treating autoimmune disorders involving an uncontrolled overproduction of IL-15.
Assuntos
Interleucina-15/imunologia , Linfócitos T/imunologia , Vacinas/imunologia , Compostos de Alúmen , Animais , Anticorpos Neutralizantes/metabolismo , Proliferação de Células , Chlorocebus aethiops , Citotoxicidade Imunológica , Humanos , Imunização , Imunogenicidade da Vacina , Interleucina-15/genética , Camundongos , Mutação/genéticaRESUMO
The emergence of SARS-CoV-2 variants, as observed with the D614G spike protein mutant and, more recently, with B.1.1.7 (501Y.V1), B.1.351 (501Y.V2) and B.1.1.28.1 (P.1) lineages, represent a continuous threat and might lead to strains of higher infectivity and/or virulence. We report on the occurrence of a SARS-CoV-2 haplotype with nine mutations including D614G/T307I double-mutation of the spike. This variant expanded and completely replaced previous lineages within a short period in the subantarctic Magallanes Region, southern Chile. The rapid lineage shift was accompanied by a significant increase of cases, resulting in one of the highest incidence rates worldwide. Comparative coarse-grained molecular dynamic simulations indicated that T307I and D614G belong to a previously unrecognized dynamic domain, interfering with the mobility of the receptor binding domain of the spike. The T307I mutation showed a synergistic effect with the D614G. Continuous surveillance of new mutations and molecular analyses of such variations are important tools to understand the molecular mechanisms defining infectivity and virulence of current and future SARS-CoV-2 strains.
Assuntos
SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Regiões Antárticas , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/genética , COVID-19/epidemiologia , COVID-19/genética , COVID-19/metabolismo , Chile , Haplótipos/genética , Humanos , Proteínas Mutantes/genética , Mutação , Ligação Proteica , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/ultraestruturaRESUMO
Vaccination development and production was an essential question for the prevention and global control of COVID-19. The strong support from governing authorities such as Operation Warp Speed and robust funding has led to the development and authorization of the tozinameran (BNT162b2) vaccine. The BNT162b2 vaccine is a lipid nanoparticle-encapsulated mRNA that encodes for SARS-CoV-2 spike protein, the main site for neutralizing antibodies. Once it binds with the host cells, the lipid nanoparticles enable the transfer of the RNA, causing S antigens' expression of the SARS-CoV-2, conferring immunity. The vaccine is administered as a 2-dose regime 21 days apart for individuals 16 years and older. Pfizer-BioNTech's BNT162b2 vaccine was the first candidate to receive FDA-Emergency Use Authorization (EUA) on December 11, 2020. During phase 2/3 clinical trials, 95% efficacy was reported among 37,706 participants over the age of 16 who received the BNT162b2 vaccination; additionally, 52% efficacy was noted 12 days following the administration of the first dose of BNT162b2, reflecting early protection of COVID-19. The BNT162b2 vaccine has exhibited 100% efficacy in clinical trials of adolescents between the ages of 12 and 15. Clinical trials in pregnant women and children under the age of 12 are expected to also exhibit promising results. This review article encompasses tozinameran (BNT162b2) vaccine journey, summarizing the BNT162b1 and BNT162b2 vaccines from preclinical studies, clinical trial phases, dosages, immune response, adverse effects, and FDA-EUA.
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Ensaios Clínicos como Assunto/métodos , Aprovação de Drogas/métodos , SARS-CoV-2/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/efeitos dos fármacos , Anticorpos Neutralizantes/metabolismo , Vacina BNT162 , COVID-19/epidemiologia , COVID-19/metabolismo , Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/metabolismo , Ensaios Clínicos como Assunto/legislação & jurisprudência , Aprovação de Drogas/legislação & jurisprudência , Avaliação Pré-Clínica de Medicamentos/métodos , Exantema/induzido quimicamente , Feminino , Humanos , Masculino , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinação/legislação & jurisprudência , Vacinação/métodosRESUMO
Dengue is a worldwide expanding threat caused by dengue virus (DENV) infection. To date, no specific treatment or effective vaccine is available. Antibodies produced by plasma cells (PCs) might be involved concomitantly in protection and severe dengue immunopathology. Although a massive appearance of PCs has been reported during acute DENV infection in humans, this response has been poorly characterized. Here, we show the dynamic of PC generation in immune-competent mice cutaneously inoculated with DENV compared with two control experimental groups: mice inoculated with inactivated DENV or with PBS. We found that PC numbers increased significantly in the skin-draining lymph node (DLN), peaking at day 10 and abruptly decreasing by day 14 after DENV inoculation. Class-switched IgG+ PCs appeared from day 7 and dominated the response, while in contrast, the frequency of IgM+ PCs decreased from day 7 onwards. Even though the kinetic of the response was similar between DENV- and iDENV-inoculated mice, the intensity of the response was significantly different. Interestingly, we demonstrated a similar PC response to virus antigens (E and prM) by ELISPOT. In situ characterization showed that PCs were distributed in the medullary cords and in close proximity to germinal centers (GCs), suggesting both an extrafollicular and a GC origin. Proliferating PCs (Ki-67+) were found as early as 3-day postinoculation, and in-depth analysis showed that these PCs were in active phases of cell cycle during the kinetic. Finally, we found a progressive appearance of high-affinity neutralizing DENV-specific IgG further supporting GC involvement. Of note, these antibodies seem to be highly cross-reactive, as a large proportion recognizes Zika virus (ZIKV). The strong PC response to skin-inoculated DENV in this work resembles the findings already described in humans. We consider that this study contributes to the understanding of the in vivo biology of the humoral immune response to DENV in an immunocompetent murine model.
Assuntos
Vírus da Dengue/imunologia , Dengue/imunologia , Plasmócitos/imunologia , Animais , Anticorpos Neutralizantes/análise , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/análise , Anticorpos Antivirais/metabolismo , Reações Cruzadas , Dengue/patologia , Dengue/virologia , Modelos Animais de Doenças , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , Masculino , Camundongos , Plasmócitos/metabolismo , Pele/imunologia , Pele/patologia , Pele/virologia , Organismos Livres de Patógenos Específicos , Zika virus/imunologiaRESUMO
The Spike protein is the target of both antibody-based therapeutics (convalescent plasma, polyclonal serum, monoclonal antibodies) and vaccines. Mutations in Spike could affect efficacy of those treatments. Hence, monitoring of mutations is necessary to forecast and readapt the inventory of therapeutics. Different phylogenetic nomenclatures have been used for the currently circulating SARS-CoV-2 clades. The Spike protein has different hotspots of mutation and deletion, the most dangerous for immune escape being the ones within the receptor binding domain (RBD), such as K417N/T, N439K, L452R, Y453F, S477N, E484K, and N501Y. Convergent evolution has led to different combinations of mutations among different clades. In this review we focus on the main variants of concern, that is, the so-called UK (B.1.1.7), South African (B.1.351) and Brazilian (P.1) strains.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , COVID-19/terapia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/metabolismo , Anticorpos Antivirais/uso terapêutico , Brasil/epidemiologia , COVID-19/epidemiologia , COVID-19/imunologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Expressão Gênica , Humanos , Evasão da Resposta Imune , Imunização Passiva/métodos , Mutação , Filogenia , Ligação Proteica , Medição de Risco , SARS-CoV-2/classificação , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , África do Sul/epidemiologia , Glicoproteína da Espícula de Coronavírus/imunologia , Reino Unido/epidemiologia , Soroterapia para COVID-19RESUMO
OBJECTIVES: The influence of glycosylation on the antigen-neutralizing ability of two potential biotherapeutic anti-human IFN-α2b antibodies composed by murine and humanized single-chain Fv fused to human Fcγ1 (chimeric and humanized scFv-Fc, respectively) was studied. RESULTS: Chimeric antibodies produced in CHO-K1 and HEK293 mammalian cells showed no differences in the antigen-antibody affinity but demonstrated differences in the in vitro neutralization of IFN-α2b activity. On the other hand, the humanized antibodies produced in the same cell types showed differences in both the antigen-antibody affinity and the antigen-neutralizing ability. These differences are due to the scFv domain, as evidenced by its expression in CHO-K1 and HEK293 cells. In order to determine if the Fc glycosylation influences the antigen binding ability, both parameters were analyzed on chimeric and humanized deglycosylated scFv-Fc. Surprisingly, no differences in the antigen-antibody affinity were observed, but differences in the antigen-neutralizing ability of both chimeric and humanized antibodies, and their respectively deglycosylated glycoforms were found. CONCLUSIONS: Fc glycosylation influences the antigen neutralization ability of two anti-rhIFN-α2b recombinant antibodies. Although affinity is the widely accepted parameter to analyze antibody antigen binding, it does not appear to be sufficient to describe the behavior of recombinant antibodies in vitro. This work contributes with a high impact knowledge to develop therapeutic recombinant antibodies where glycosylation and producer cell lines must be taken into account for their influence on the antigen binding capacity and not only for their impact on the effector properties as it has been historically considered for antibodies.
Assuntos
Anticorpos Neutralizantes , Interferon-alfa/imunologia , Proteínas Recombinantes , Anticorpos de Cadeia Única , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Afinidade de Anticorpos , Células CHO , Cricetinae , Cricetulus , Glicosilação , Células HEK293 , Humanos , Interferon alfa-2 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismoRESUMO
Background: Gardnerella vaginalis is a bacterial vaginosis (BV)-associated vaginal bacterium that produces the toxin vaginolysin (VLY). VLY is a pore-forming toxin that is suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and the emergence of antibiotic-resistant bacterial species demonstrate the need for the development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve the properties of scFvs, monospecific dimeric scFvs were generated by the genetic fusion of two anti-VLY scFv molecules connected by an alpha-helix-forming peptide linker. Results: N-terminal hexahistidine-tagged dimeric scFvs were constructed and produced in Escherichia coli and purified using metal chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by in vitro hemolytic assay. The circulating half-life of purified scFvs in the blood plasma of mice was determined by ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life than parental monomeric scFv. Conclusions: The protein obtained by the genetic fusion of two anti-VLY scFvs into a dimeric molecule exhibited improved properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by the bacteria G. vaginalis.