RESUMO
Salmonella Typhimurium is the most prevalent non-host specific Salmonella serovars and a major concern for both human and animal health systems worldwide contributing to significant economic loss. Type 3 secretion system (T3SS) of Salmonella plays an important role in bacterial adherence and entry into the host epithelial cells. The product of invH gene of Salmonella is an important component of the needle complex of the type 3 secretion system. Hence, the present study was undertaken to clone and express the 15 kDa InvH surface protein of Salmonella Typhimurium in an E. coli host and to evaluate its immune potency in mice. The purified recombinant InvH (r-InvH) protein provoked a significant (p < 0.01) rise in IgG in the inoculated mice. The immunized mice were completely (100%) protected against the challenge dose of 107.5 LD50, while protection against challenge with the same dose of heterologous serovars was 90%. The bacterin-vaccinated group showed homologous protection of 60% against all three serovars. Findings in this study suggest the potential of the r-InvH protein of S. Typhimurium as an effective vaccine candidate against Salmonella infections.
Assuntos
Intoxicação Alimentar por Salmonella , Salmonelose Animal , Infecções por Salmonella , Animais , Camundongos , Humanos , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Escherichia coli/genética , Proteínas de Bactérias/metabolismo , Infecções por Salmonella/prevenção & controle , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/metabolismo , Salmonelose Animal/microbiologia , Vacinas AtenuadasRESUMO
The Zika Virus (ZIKV) is an emerging arbovirus of great public health concern, particularly in the Americas after its last outbreak in 2015. There are still major challenges regarding disease control, and there is no ZIKV vaccine currently approved for human use. Among many different vaccine platforms currently under study, the recombinant envelope protein from Zika Virus (rEZIKV) constitutes an alternative option for vaccine development and has great potential for monitoring ZIKV infection and antibody response. This study describes a method to obtain a bioactive and functional rEZIKV using an E. coli expression system, with the aid of a 5-L airlift bioreactor and following an automated fast protein liquid chromatography (FPLC) protocol, capable of obtaining high yields of approximately 20 mg of recombinant protein per liter of bacterium cultures. The purified rEZIKV presented preserved antigenicity and immunogenicity. Our results show that the use of an airlift bioreactor for the production of rEZIKV is ideal for establishing protocols and further research on ZIKV vaccines bioprocess, representing a promising system for the production of a ZIKV envelope recombinant protein-based vaccine candidate.
Assuntos
Vacinas Virais , Infecção por Zika virus , Zika virus , Humanos , Zika virus/genética , Proteínas do Envelope Viral/genética , Anticorpos Neutralizantes , Escherichia coli , Anticorpos Antivirais , Vacinas Virais/genética , Vacinas de Subunidades Antigênicas/genética , Proteínas Recombinantes/genética , Reatores BiológicosRESUMO
COVID-19 pandemic poses a serious threat to human health; it has completely disrupted global stability, making vaccine development an important goal to achieve. Monoclonal antibodies play an important role in subunit vaccines strategies. In this work, nine murine MAbs against the RBD of the SARS-CoV-2 spike protein were obtained by hybridoma technology. Characterization of purified antibodies demonstrated that five of them have affinities in the order of 108 L/mol. Six MAbs showed specific recognition of different recombinant RBD-S antigens in solution. Studies of the additivity index of anti-RBD antibodies, by using a novel procedure to determine the additivity cut point, showed recognition of at least five different epitopes. The MAbs CBSSRBD-S.11 and CBSSRBD-S.8 revealed significant neutralizing capacity against SARS-CoV-2 in an ACE2-RBD binding inhibition assay (IC50 = 85.5pM and IC50 = 122.7pM, respectively) and in a virus neutralizing test with intact SARS-CoV-2 (VN50 = 0.552 nM and VN50 = 4.854 nM, respectively) when D614G strain was used to infect Vero cells. Also CBSSRBD-S.11 neutralized the SARS-CoV-2 strains Alpha and Beta: VN50 = 0.707 nM and VN50 = 0.132 nM, respectively. The high affinity CBSSRBD-S.8 and CBSSRBD-S.7 recognized different epitopes, so they are suitable for the development of a sandwich ELISA to quantitate RBD-S recombinant antigens in biomanufacturing processes, as well as in pharmacokinetic studies in clinical and preclinical trials.
Assuntos
Anticorpos Monoclonais/metabolismo , Vacinas contra COVID-19/imunologia , COVID-19/diagnóstico , SARS-CoV-2/fisiologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/genética , COVID-19/imunologia , Vacinas contra COVID-19/genética , Ensaios Clínicos como Assunto , Feminino , Engenharia Genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Domínios e Motivos de Interação entre Proteínas/genética , Desenvolvimento de Vacinas , Vacinas de Subunidades Antigênicas/genéticaRESUMO
Haemophilus influenzae is a common organism of the human upper respiratory tract; this bacterium is responsible of a wide spectrum for respiratory infections and can generate invasive diseases such as meningitis and septicemia. These infections are associated with H. influenzae encapsulated serotype b. However, the incidence of invasive disease caused by nontypeable H. influenzae (NTHi) has increased in the post-H. influenzae serotype b (Hib) vaccine era. Currently, an effective vaccine against NTHi is not available; due to this, it is important to find an antigen capable to confer protection against NTHi infection. In this study, 10 linear B cell epitopes and 13 CTL epitopes and a putative plasminogen-binding motif (252FYNKENGMY260) and the presence of enolase on the surface of different strains of H. influenzae were identified in the enolase sequence of H. influenzae. Both in silico and experimental results showed that recombinant enolase from H. influenzae is immunogenic that could induce a humoral immune response; this was observed mediating the generation of specific polyclonal antibodies anti-rNTHiENO that recognize typeable and nontypeable H. influenzae strains. The immunogenic properties and the superficial localization of enolase in H. influenzae, important characteristics to be considered as a new candidate for the development of a vaccine, were demonstrated.
Assuntos
Proteínas de Bactérias/imunologia , Infecções por Haemophilus/prevenção & controle , Vacinas Anti-Haemophilus/imunologia , Haemophilus influenzae/imunologia , Fosfopiruvato Hidratase/imunologia , Infecções Respiratórias/prevenção & controle , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Biologia Computacional , Epitopos/genética , Epitopos/imunologia , Infecções por Haemophilus/imunologia , Infecções por Haemophilus/microbiologia , Vacinas Anti-Haemophilus/genética , Vacinas Anti-Haemophilus/uso terapêutico , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Humanos , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Infecções Respiratórias/imunologia , Infecções Respiratórias/microbiologia , Desenvolvimento de Vacinas , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêuticoAssuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sinciciais Respiratórios/fisiologia , Adenoviridae/genética , Animais , Ensaios Clínicos como Assunto , Humanos , Imunidade Celular , Imunidade Humoral , Vacinas Atenuadas , Vacinas de Subunidades Antigênicas/genética , Vacinas Sintéticas/genética , Proteínas Virais de Fusão/genéticaRESUMO
Leptospirosis is a zoonosis of worldwide distribution, caused by infection with pathogenic Leptospira species. The vaccines that are currently available are bacterins, with limited human use, that confer short-term, serovar-specific immunity. Lig proteins are considered to be the best vaccine candidates to date. Here, we aimed to construct a recombinant Lig chimera (LC) comprised of LigAni and LigBrep fragments, and to evaluate it as subunit or DNA vaccine using different administration strategies. Vaccines were formulated with 50⯵g of recombinant LC associated with different adjuvants or with 100⯵g of pTARGET/LC. Four-week-old hamsters received two doses of vaccine with different strategies and were challenged with 5â¯×â¯DL50Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. The immune response generated by Lig chimera conferred 100% protection to hamsters treated with at least one dose of recombinant LC. Despite the high levels of antibodies that vaccinated animals produced, a sterilizing immunity was not achieved. The lack of a sterilizing immunity could indicate the importance of a mixed humoral and cellular immune response. The present study generated insights that will be useful in the future development of improved subunit vaccines against leptospirosis.
Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Leptospira/imunologia , Leptospirose/prevenção & controle , Proteínas Recombinantes de Fusão/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Vacinas Bacterianas/genética , Células CHO , Cricetinae , Cricetulus , Expressão Gênica , Humanos , Leptospira/genética , Leptospirose/imunologia , Leptospirose/microbiologia , Leptospirose/mortalidade , Proteínas Recombinantes de Fusão/genética , Vacinas de DNA , Vacinas de Subunidades Antigênicas/genéticaRESUMO
Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.
Assuntos
Epitopos de Linfócito B/imunologia , Imunogenicidade da Vacina , Vacinas Antimaláricas/imunologia , Proteínas de Membrana/imunologia , Peptídeos/síntese química , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Formação de Anticorpos , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulina G/imunologia , Vacinas Antimaláricas/genética , Malária Vivax/prevenção & controle , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Peptídeos/imunologia , Plasmodium vivax , Proteínas de Protozoários/genética , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/imunologia , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Due to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.
Assuntos
Vacinas contra Leishmaniose/imunologia , Leishmaniose/prevenção & controle , Vacinas de Subunidades Antigênicas/imunologia , Adjuvantes Imunológicos , Animais , Antígenos de Protozoários/imunologia , Ensaios Clínicos como Assunto , Humanos , Leishmaniose/imunologia , Vacinas contra Leishmaniose/genética , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/prevenção & controle , Camundongos , Mapeamento de Peptídeos , Vacinação , Vacinas de Subunidades Antigênicas/genéticaRESUMO
Our group has developed a subunit vaccine candidate against Dengue virus (DENV) based on two different viral regions, the domain III of the envelope protein and the capsid protein. The chimeric proteins for each serotype (DIIIC1-4), aggregated with the oligodeoxynucleotide 39 M, form the tetravalent formulation named Tetra DIIIC. Tetra DIIIC induces a protective immune response in mice when it is inoculated by intraperitoneal route. However, if children are the main targets for a DENV vaccine, then a needle-free route of administration should be attractive and advantageous. In this study, we evaluated for the first time, in vivo, a vaccine candidate against DENV based on recombinant proteins using the intranasal route. After three doses of Tetra DIIIC in mice, we measured the humoral immune response against the four DENV serotypes and the corresponding recombinant proteins. Moreover, the functionality of these antibodies was evaluated through a plaque reduction neutralization test. Finally, to assess the cellular immune response induced, we measured the IFN-γ-levels secreted by spleen cells after in vitro stimulation with DENV. The results presented in this study indicate that the intranasal immunization with Tetra DIIIC favors the generation of DENV-specific cell-mediated immunity. On the other hand, the immunization using intraperitoneal and intranasal routes, simultaneously, generate functional antibodies (anti-DIIIC and anti-DENV) and an in vitro response of IFN-γ secretion.
Assuntos
Vírus da Dengue/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Administração Intranasal , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Testes de Neutralização , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/imunologia , Ensaio de Placa Viral , Vacinas Virais/genéticaRESUMO
Streptococcus uberis is one of the most prevalent pathogens causing clinical and subclinical mastitis worldwide. Among bacterial factors involved in intramammary infections caused by this organism, S. uberis adhesion molecule (SUAM) is one of the main virulence factors identified. This molecule is involved in S. uberis internalization to mammary epithelial cells through lactoferrin (Lf) binding. The objective of this study was to evaluate SUAM properties as a potential subunit vaccine component for prevention of S. uberis mastitis. B epitope prediction analysis of SUAM sequence was used to identify potentially immunogenic regions. Since these regions were detected all along the gene, this criterion did not allow selecting a specific region as a potential immunogen. Hence, four fractions of SUAM (-1fr, 2fr, 3fr and 4fr), comprising most of the protein, were cloned and expressed. Every fraction elicited a humoral immune response in mice as predicted by bioinformatics analysis. SUAM-1fr generated antibodies with the highest recognition ability towards SUAM native protein. Moreover, antibodies against SUAM-1fr produced the highest proportion of internalization inhibition of S. uberis to mammary epithelial cells. In conclusion, SUAM immunogenic and functionally relevant regions were identified and allowed to propose SUAM-1fr as a potential candidate for a subunit vaccine for S. uberis mastitis prevention.