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1.
Biomedicines ; 11(11)2023 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-38002050

RESUMO

In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein.

2.
Antibiotics (Basel) ; 12(10)2023 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-37887185

RESUMO

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a multifunctional neuropeptide that is widely distributed and conserved across species. We have previously shown that in teleost fish, PACAP not only possesses direct antimicrobial properties but also immunomodulatory effects against the bacterial pathogens Flavobacterium psychrophilum and Pseudomonas aeruginosa using in vitro and in vivo experiments. These previous results suggest PACAP can be used as an alternative to antibiotics to prevent and/or treat bacterial infections in the aquaculture industry. To accomplish this goal, more studies are needed to better understand the effect of PACAP on pathogens affecting fish in live infections. In the present study, the transcripts PACAP, PRP/PACAP, and VPAC2 receptor were examined in rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri, which exhibited an increase in their expression in the spleen when compared to healthy fish. Synthetic Clarias gariepinus PACAP-38 has direct antimicrobial activity on Y. ruckeri and inhibits up to 60% of the bacterial growth when the peptide is at concentrations between 50 and 100 µM in TSB. The growth inhibition increased up to 90% in the presence of 12.5 µM of PACAP-38 when salt-free LB broth was used instead of TSB. It was also found to inhibit Y. ruckeri growth in a dose-dependent manner when the rainbow trout monocyte/macrophage-like cell line (RTS11) was pre-treated with lower concentrations of the peptide (0.02 and 0.1 µM) before going through infection. Differential gene expression was analyzed in this in vitro model. Overall, the results revealed new evidence to support the role of PACAP as an antimicrobial and immunomodulatory peptide treatment in teleosts.

3.
PLoS One ; 18(9): e0288006, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37751460

RESUMO

Despite that more than one hundred vaccines against SARS-CoV-2 have been developed and that some of them were evaluated in clinical trials, the latest results revealed that these vaccines still face great challenges. Among the components of the virus, the N-protein constitutes an attractive target for a subunit vaccine because it is the most abundant, highly conserved and immunogenic protein. In the present work, a chimeric protein (N-CD protein) was constructed by the fusion of the N-protein to the extracellular domain of human CD154 as the molecular adjuvant. HEK-293 cells were transduced with lentiviral vector bearing the N-CD gene and polyclonal cell populations were obtained. The N-CD protein was purified from cell culture supernatant and further characterized by several techniques. Immunogenicity studies in mice and non-human primates showed the N-CD protein induced high IgG titers in both models after two doses. Moreover, overall health monitoring of non-human primates demonstrated that animals were healthy during 228 days after first immunization. Data obtained support further investigation in order to develop this chimeric protein as vaccine candidate against COVID-19 and other coronavirus diseases.


Assuntos
COVID-19 , Vacinas , Humanos , Animais , Camundongos , SARS-CoV-2/genética , COVID-19/prevenção & controle , Células HEK293 , Vacinas contra COVID-19 , Nucleocapsídeo , Ligante de CD40/genética , Proteínas Recombinantes de Fusão/genética
4.
Fish Shellfish Immunol Rep ; 4: 100093, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37122444

RESUMO

Teleost IgT/Z plays a principal role in the defense mechanisms against infectious agents in the mucosal compartments and in systemic immunity. Previously, Nile tilapia (Oreochromis niloticus) IgT was discovered and characterized at transcription level. In this work, we generated a monoclonal antibody (mAb) that specifically recognized the Nile tilapia IgT. BALB/c mice were immunized with three synthetic peptides conjugated to KLH. The sequences of these peptides derived from the constant region of the Nile tilapia IgT heavy chain. ELISA and Western blotting confirmed the specificity of the polyclonal sera and the culture supernatant from a positive hybridoma clone. We observed immunoreactivity against a recombinant IgT fragment and native IgT in skin mucus. The anti-IgT mAb did not cross-react with purified tilapia IgM. Direct ELISA analysis allowed the quantification of skin mucus IgM and IgT concentrations. Flow cytometry analysis revealed differences in the percentage of IgT+ B cell populations between juveniles and adults in peripheral blood, head kidney and spleen lymphocytes and among the tissues analyzed. For further validation of the anti-IgT mAb utility, a recombinant vaccine candidate against sea lice (TT-P0 Ls) was injected into juvenile tilapia. Direct ELISA results revealed a differential secretion of skin mucus IgT and IgM after immunostimulation. In addition, the percentages of IgT+ B cells were determined at 7 days after booster and ex-vivo stimulation by flow cytometry. This mAb constitutes an important immunological tool to study the biological function and structural characteristics of tilapia IgT.

5.
Vaccines (Basel) ; 10(6)2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35746505

RESUMO

COVID-19 is a respiratory viral disease caused by a new coronavirus called SARS-CoV-2. This disease has spread rapidly worldwide with a high rate of morbidity and mortality. The receptor-binding domain (RBD) of protein spike (S) mediates the attachment of the virus to the host's cellular receptor. The RBD domain constitutes a very attractive target for subunit vaccine development due to its ability to induce a neutralizing antibody response against the virus. With the aim of boosting the immunogenicity of RBD, it was fused to the extracellular domain of CD154, an immune system modulator molecule. To obtain the chimeric protein, stable transduction of HEK-293 was carried out with recombinant lentivirus and polyclonal populations and cell clones were obtained. RBD-CD was purified from culture supernatant and further characterized by several techniques. RBD-CD immunogenicity evaluated in mice and non-human primates (NHP) indicated that recombinant protein was able to induce a specific and high IgG response after two doses. NHP sera also neutralize SARS-CoV-2 infection of Vero E6 cells. RBD-CD could improve the current vaccines against COVID-19, based in the enhancement of the host humoral and cellular response. Further experiments are necessary to confirm the utility of RBD-CD as a prophylactic vaccine and/or booster purpose.

6.
Fish Shellfish Immunol ; 115: 150-159, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34146673

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide that belongs to the secretin/glucagon/GHRH/VIP superfamily. Some of these molecules have antimicrobial activity and they are capable of stimulating the immune system. The present work studied the antibacterial and immunostimulatory activity of PACAP-38 from African catfish Clarias gariepinus against the Gram-negative bacterium Pseudomonas aeruginosa in an in vivo test. PACAP-38 improved antimicrobial activity of skin mucus molecules against P. aeruginosa. The peptide modulates the gene expression profile of TLR-1, TLR-5, MyD88, IL-1ß, TNF-ɑ, IL-8, pardaxin, hepcidin and G/C-type lysozymes in skin, spleen and head kidney. The influenced exerted depended on the time after infection and tissue analyzed. This study provides the first evidence of a link between PACAP and antimicrobial peptides hepcidin and pardaxin. Our results suggest further use of PACAP as antimicrobial agent that could potentially be used to control disease in aquaculture.


Assuntos
Anti-Infecciosos/imunologia , Peixes-Gato/genética , Peixes-Gato/imunologia , Proteínas de Peixes/genética , Imunidade Inata/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Transdução de Sinais/genética , Animais , Proteínas de Peixes/imunologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Transdução de Sinais/imunologia , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/imunologia , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/imunologia
7.
Fish Shellfish Immunol ; 110: 44-54, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33348037

RESUMO

Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide-based strategy. The peptide sequence was selected from the surface-exposed region between CH3-CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.


Assuntos
Anticorpos Monoclonais/imunologia , Ciclídeos/imunologia , Proteínas de Peixes/imunologia , Imunidade Humoral , Cadeias Pesadas de Imunoglobulinas/imunologia , Imunoglobulina M/imunologia , Sequência de Aminoácidos , Animais , Alinhamento de Sequência
8.
Fish Shellfish Immunol ; 103: 58-65, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32334130

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide belonging to the glucagon/secretin superfamily. In teleost fish, PACAP has been demonstrated to have an immunomodulatory role. Although previous studies have shown that viral/bacterial infections can influence the transcription of PACAP splicing variants and associated receptors in salmonids, the antiviral activity of PACAP has never been studied in teleost. Thus, in the present work, we investigated in vitro the influence of synthetic Clarias gariepinus PACAP-38 on the transcription of genes related to viral immunity using the rainbow trout monocyte/macrophage-like cell line RTS11 as a model. Positive transcriptional modulation of interferon gamma (IFNγ), interferon alpha (FNα1,2), interleukin 8 (IL-8), Mx and Toll-like receptor 3 (TLR3) genes was found in a dose and time dependent manner. We also explored how a pre-treatment with PACAP could enhance antiviral immune response using poly (I:C) as viral mimic. Interferons and IL-8 transcription levels were enhanced when PACAP was added 24 h previous to poly (I:C) exposure. With these evidences, we tested in vivo how PACAP administration by immersion bath affected the survival of rainbow trout fry to a challenge with viral hemorrhagic septicemia virus (VHSV). After challenge, PACAP-treated fish had increased survival compared to non-treated/challenge fish. Furthermore, PACAP was able to decrease the viral load in spleen/kidney and stimulate the transcription of IFNs and Mx when compared to untreated infected fish. Altogether, the results of this work provide valuable insights regarding the role of teleost PACAP in antiviral immunity and point to a potential application of this peptide to reduce the impact of viral infections in aquaculture.


Assuntos
Antivirais/imunologia , Peixes-Gato/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Imunidade Inata , Oncorhynchus mykiss , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Animais , Proteínas de Peixes/imunologia , Novirhabdovirus/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Poli I-C/farmacologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária
9.
Fish Shellfish Immunol ; 92: 322-330, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31200071

RESUMO

The development of vaccines employing conserved protein antigens, for instance ribosomal protein P0, has as disadvantage the high degree of identity between pathogen and host proteins due to possible induction of tolerance or auto antibodies in the host organism. To overcome this drawback, peptide-based vaccines have been designed with a proved high efficacy. The use of defined peptides as antigens has the problem that they are generally poor immunogenic unless coupled to a carrier protein. Several studies have established the potential for promiscuous T cell epitopes incorporated into chimeric peptides to enhance the immunogenicity in mammals. On the contrary, studies about the role of these epitopes on teleost immune system are scarce. Therefore, the main objective of our present study was to evaluate the potential of promiscuous T cell epitopes to boost specific IgM immune response in teleost fish against a peptide antigen. With this aim, we used a peptide of 35 amino acids from the ribosomal P0 protein of Lepeophtheirus salmonis, an important parasite in salmon aquaculture. We fused this peptide to the C-terminal of T cell epitopes from tetanus toxin and measles virus and produced the chimeric protein in Escherichia coli. Following vaccination, IgM antibody production was monitored in different immunization schemes in Tilapia, African catfish and Atlantic salmon. The results demonstrated for first time that the addition of T cell epitopes at the N-terminal of a target peptide increased IgM specific response in different teleost species, revealing the potential of this approach to develop peptide-based vaccines for aquaculture. The results are also of great importance in the context of vaccine development against sea lice using ribosomal protein P0 as antigen taking into account the key role of P0 in protein synthesis and other essential physiological processes.


Assuntos
Copépodes/imunologia , Ectoparasitoses/veterinária , Epitopos de Linfócito T/imunologia , Doenças dos Peixes/imunologia , Imunidade Inata/efeitos dos fármacos , Imunoglobulina M/imunologia , Animais , Proteínas de Artrópodes/imunologia , Peixes-Gato/imunologia , Ciclídeos/imunologia , Ectoparasitoses/imunologia , Peptídeos/imunologia , Proteínas Ribossômicas/imunologia , Salmo salar/imunologia , Vacinas de Subunidades Antigênicas/imunologia
10.
Dev Comp Immunol ; 88: 124-136, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30012536

RESUMO

Immunoglobulin molecules play an important role in the immune defense system in all jawed vertebrates, by protecting the organism from a wide variety of pathogens. Nile tilapia (Oreochromis niloticus) is extensively cultivated worldwide, with a strong established market demand. It constitutes one of the model species for the study of fish immunology and its genome is currently fully sequenced. The presence of the immunoglobulin M gene in this species is well documented, as well as its major role in systemic immunity. To date, the IgT gene from O. niloticus has not been identified and, therefore, no information is available on the role of this immunoglobulin isotype in the immune response in tilapia. In the present work, novel secreted and membrane immunoglobulin T isotypes and a fragment of IgM were isolated from tilapia head kidney lymphocytes. Their transcriptional profiles were analyzed by quantitative PCR in larval development and in different tissues of healthy or lipopolysaccharide/Edwardsiella tarda-challenged tilapia adults. The presence of IgT and IgM were detected in early stages of larval development. Additionally, these genes exhibited differential expression profiles in basal conditions and after E. tarda infection in adult tilapia, in accord with the proposed effector functions of these immunoglobulins in the systemic and mucosal compartments. Our results suggest the potential involvement of this new Ig in mucosal immunity in tilapia.


Assuntos
Ciclídeos/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Imunidade nas Mucosas , Imunoglobulinas/imunologia , Animais , Biomarcadores , Edwardsiella tarda/imunologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Infecções por Enterobacteriaceae/virologia , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Proteínas de Peixes/isolamento & purificação , Perfilação da Expressão Gênica , Rim Cefálico/citologia , Imunoglobulinas/genética , Imunoglobulinas/isolamento & purificação , Larva/imunologia , Lipopolissacarídeos/imunologia , Linfócitos/metabolismo , Filogenia
11.
Fish Shellfish Immunol ; 71: 275-285, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29017941

RESUMO

Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. This cytokine plays a very important role in defining Th1 immune response in all vertebrates. In the present study, we identified and isolated for the first time the gene coding for Nile tilapia (Oreochromis niloticus) IFNγ from spleen lymphocytes. The isolated tilapia IFNγ has between 24 and 62% of amino acid identity as compared to reported sequences for other teleost fishes. It has close phylogenetic relationships with IFNγ molecules belonging to the group of Perciforms and presents the typical structural characteristics of gamma interferon molecules. The tissue expression analysis showed that IFNγ is expressed constitutively in head kidney, skin, intestine, muscle and brain. Its expression was not detected in gills by conventional RT-PCR. However, under conditions of stimulation with Poly I:C and LPS, IFNγ expression was up-regulated in gills after 24 h post-stimulation. IFNγ expression was also induced in gills 24 h after Edwardsiella tarda infection suggesting its important role in immunity against intracellular bacteria. The recombinant protein produced in Escherichia coli induced Mx gene transcription in head kidney primary culture cells. These results are the first steps to characterize the role of tilapia IFNγ in the defense against pathogens in tilapia. Furthermore, the isolation of this molecule provides a new tool to characterize the cellular immune response to various stimuli in this organism.


Assuntos
Ciclídeos/genética , Ciclídeos/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Interferon gama/genética , Interferon gama/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Interferon gama/química , Lipopolissacarídeos/farmacologia , Filogenia , Poli I-C/farmacologia , Alinhamento de Sequência/veterinária
12.
Vaccine ; 35(42): 5722-5728, 2017 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-28893476

RESUMO

Modern subunit vaccines have excellent safety profiles and improved tolerability, but do not elicit strong immune responses without the addition of adjuvants. Developing a safe and affective adjuvant remains a challenge for peptide-based vaccine design. Growth Hormone Releasing Peptide-6 (GHRP-6) is one of the earliest-developed, synthetic, peptidyl growth hormone secretagogue receptor agonists. These compounds mimic the effect of the endogenous ligand, ghrelin. In the present study, we evaluated the ability of GHRP-6 to enhance the humoral immune response against co-injected antigens in mice, tilapia and African catfish. This peptide was able to increase the antigen-specific antibody response using heterologous proteins and peptides as antigens, which were also formulated in "water in oil" emulsions (Freund and Montanide). As long as we know there is no previous report describing any ghrelin analogous as molecular immunomodulator stimulating a humoral immune response. Further studies will be conducted to evaluate the functionality of this humoral immune response in challenge trials.


Assuntos
Anticorpos/imunologia , Peixes-Gato/imunologia , Ciclídeos/imunologia , Oligopeptídeos/imunologia , Tilápia/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos/imunologia , Feminino , Grelina/imunologia , Imunidade Humoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas de Subunidades Antigênicas/imunologia
13.
Vaccine ; 35(34): 4437-4443, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28688785

RESUMO

Classical swine fever is an economically important, highly contagious disease of swine worldwide. Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, a lentivirus-based gene delivery system is used to obtain a stable recombinant HEK 293 cell line for the expression of E2-CSFV antigen fused to porcine CD154 as immunostimulant molecule. The E2-CD154 chimeric protein was secreted into the medium by HEK293 cells in a concentration around 50mg/L in suspension culture conditions using spinner bottles. The E2-CD154 immunized animals were able to overcome the challenge with a high virulent CSF virus strain performed 7days after a unique dose of the vaccine without clinical manifestations of the disease. Specific anti-CSFV neutralizing antibodies and IFN-γ were induced 8days after challenge equivalent to 14days post-vaccination. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after a single vaccination. These results suggest that the E2-CD154 antigen could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.


Assuntos
Ligante de CD40/imunologia , Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Peste Suína Clássica/imunologia , Células HEK293 , Humanos , Lentivirus/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Suínos , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/administração & dosagem
14.
Fish Shellfish Immunol ; 50: 50-5, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26804662

RESUMO

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In previous studies, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide in vitro e in vivo. Hyperimmune serum was obtained from rabbits immunized with a P. argus -NOS fragment of 31 kDa produced in Escherichia coli, which specifically detected the recombinant polypeptide and the endogenous NOS from lobster hemocytes by western blotting and immunofluorescence. In the present work, we demonstrate that the hyperimmune serum obtained against P. argus NOS also recognizes Litopenaeus vannamei NOS in hemocytes by western blotting and immunofluorescence. Our data also show that while the hemolymph of L. vannamei has a strong antibacterial activity against the Gram negative bacteria Aeromonas hydrophila, the administration of the anti NOS serum reduce the natural bacterial clearance. These results strongly suggest that NOS is required for the shrimp immune defense toward Gram negative bacteria. Therefore, the monitoring of induction of NOS could be an important tool for testing immunity in shrimp farming.


Assuntos
Aeromonas hydrophila/fisiologia , Proteínas de Artrópodes/metabolismo , Imunidade Inata , Óxido Nítrico Sintase/metabolismo , Penaeidae/genética , Penaeidae/imunologia , Animais , Anti-Infecciosos/metabolismo , Hemolinfa/imunologia , Penaeidae/microbiologia
15.
Vaccine ; 32(2): 223-9, 2014 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-24252704

RESUMO

Modern vaccines based on purified recombinant antigens have improved their safety; however they induce a suboptimal immune response without the help of adjuvants. Consequently, the development of new adjuvants to enhance the immunogenicity of purified subunit antigens and modulate resulting immune responses is of great interest. In the present study, we evaluated the ability of antimicrobial peptides Oreochromicins previously isolated from tilapia Oreochromis niloticus to enhance adaptive immune responses in mice and tilapia. When co-administrated with ovalbumin in mice, Oreochromicin-1 induced a TH1 humoral immune response. Oreochromicin-2 and 3 induce a TH1 cellular immune response characterized by the induction of interferon-γ in a dose depend manner. Additionally, co-administration of Oreochromicin-1 with the sea lice my32 from Lepeophtheirus salmonis antigen (my32-Ls) increases the humoral immune response in mice and tilapia. We also tested different combinations of these Oreochromicins with the sea lice antigen my32-Ls in mice. Humoral and cellular TH1 responses were enhanced by co-administration of my32-Ls/Oreochromicin-3 and the combination my32-Ls/Oreochromicin-2/3. In agreement with these results, Oreochromicin-1 and 3 enhanced in vitro TH1 cytokine IFN-γ production in Concanavalin A primed splenocytes from naïve mice after a 48h incubation period. In summary, the results showed that tilapia alpha-helical antimicrobial peptides Oreochromicins are able to boost immune response in mammals and fish, encouraging their use as TH1 molecular adjuvants to subunit antigens.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Ciclídeos/imunologia , Células Th1/imunologia , Vacinas/imunologia , Imunidade Adaptativa , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos/imunologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Células Cultivadas , Copépodes , Feminino , Imunidade Celular , Imunidade Humoral , Interferon gama/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologia
16.
Fish Shellfish Immunol ; 35(6): 1788-96, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24036332

RESUMO

The high conservation of the pituitary adenylate cyclase activating polypeptide (PACAP) sequence indicates that this peptide fulfills important biological functions in a broad spectrum of organisms. However, in invertebrates, little is known about its presence and its functions remain unclear. Up to now, in non-mammalian vertebrates, the majority of studies on PACAP have focused mainly on the localization, cloning and structural evolution of this peptide. As yet, little is known about its biological functions as growth factor and immunomodulator in lower vertebrates. Recently, we have shown that PACAP, apart from its neuroendocrine role, influences immune functions in larval and juvenile fish. In this work, we isolated for the first time the cDNA encoding the mature PACAP from a crustacean species, the white shrimp Litopenaeus vannamei, corroborating its high degree of sequence conservation, when compared to sequences reported from tunicates to mammalian vertebrates. Based on this, we have evaluated the effects of purified recombinant Clarias gariepinus PACAP administrated by immersion baths on white shrimp growth and immunity. We demonstrated that PACAP improves hemocyte count, superoxide dismutase, lectins and nitric oxide synthase derived metabolites in treated shrimp related with an increase in total protein concentration and growth performance. From our results, PACAP acts as a regulator of shrimp growth and immunity, suggesting that in crustaceans, as in vertebrate organisms, PACAP is an important molecule shared by both the endocrine and the immune systems.


Assuntos
Proteínas de Artrópodes/genética , Penaeidae/genética , Penaeidae/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli/genética , Larva/genética , Larva/crescimento & desenvolvimento , Larva/imunologia , Dados de Sequência Molecular , Penaeidae/crescimento & desenvolvimento , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Reação em Cadeia da Polimerase/veterinária , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/veterinária
17.
Exp Parasitol ; 135(2): 188-99, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23850998

RESUMO

Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry. Vaccination could be an environmentally friendly alternative for sea lice control; however, research on the development of such vaccines is still at an early stage of development. Recent results have suggested that subolesin/akirin/my32 are good candidate antigens for the control of arthropod infestations, including sea lice, but background knowledge about these genes in crustaceans is limited. Herein, we characterize the my32 gene/protein from two important sea lice species, Caligus rogercresseyi and Lepeophtheirus salmonis, based on cDNA sequence isolation, phylogenetic relationships, three dimensional structure prediction and expression analysis. The results show that these genes/proteins have the main characteristics of akirins from invertebrates. In addition, immunization with purified recombinant my32 from L. salmonis elicited a specific antibody response in mice and fish. These results provide an improvement to our current knowledge about my32 proteins and their potential use as vaccine candidates against sea lice in fish.


Assuntos
Antígenos/imunologia , Copépodes/imunologia , Doenças dos Peixes/prevenção & controle , Salmo salar/parasitologia , Vacinas , Sequência de Aminoácidos , Animais , Formação de Anticorpos , Antígenos/química , Antígenos/genética , Aquicultura , Sequência de Bases , Chile , Clonagem Molecular , Copépodes/química , Copépodes/genética , DNA Complementar/química , Feminino , Doenças dos Peixes/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Noruega , Filogenia , Conformação Proteica , RNA/genética , RNA/isolamento & purificação , Alinhamento de Sequência , Tilápia
18.
Nitric Oxide ; 25(4): 396-406, 2011 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-21971212

RESUMO

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In a previous study, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide (LPS) in vitro. In the present work, lobster hemocytes and gills exposed to Escherichia coli O55:B5 LPS showed an increase in both NOS activity and NOS gene expression in vivo. This response was dose and time dependent. The 3D NOS structure was predicted by comparative modeling showing the oxygenase and reductase domains. These domains contain the conserved binding motifs of NOS already found in a variety of organisms. The 3D structure prediction analysis allowed the selection of a fragment of 666bp that was cloned and subsequently expressed in E. coli BL21, in which a recombinant product of around 31KDa was obtained. Hyperimmune serum obtained from immunized rabbits was tested and employed to specifically detect the recombinant polypeptide or the endogenous NOS from lobster hemocytes by western blot and immunofluorescence. This study contributes to enlarge the existing knowledge related to NOS structure and NOS participation in the immune response in lobsters. The evaluation of an antibody capable to recognize NOS from lobsters constitutes a novel and interesting tool for the implementation of further studies on NOS functions in crustaceans.


Assuntos
Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase/metabolismo , Palinuridae/enzimologia , Palinuridae/imunologia , Sequência de Aminoácidos , Animais , Western Blotting , Clonagem Molecular , Relação Dose-Resposta Imunológica , Ativação Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Imunofluorescência , Brânquias/citologia , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Hemócitos/citologia , Hemócitos/efeitos dos fármacos , Hemócitos/enzimologia , Soros Imunes , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/imunologia , Palinuridae/genética , Conformação Proteica , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo
19.
Vaccine ; 29(15): 2810-20, 2011 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-21320542

RESUMO

Sea lice (Copepoda, Caligidae) are the most widely distributed marine pathogens in the salmon industry in the last 30 years. Caligus rogercresseyi is the most important species affecting Chile's salmon industry. Vaccines against caligid copepods have the potential to be a cost-effective means of controlling the infestation and avoid many of the disadvantages of medicine treatments. However, research in the development of such vaccines has begun only recently and approaches used thus far have met with little or no success. In the present study, we characterized a novel gene (denoted as my32) from C. rogercresseyi which has the highest identity with the Lepeophtheirus salmonis gene akirin-2. To assess the function of the gene an RNA interference experiment was developed and a reduction in the number of ectoparasites on fish in the my32-dsRNA treated group was observed. The recombinant my32 protein was used in a vaccination-challenge trial to evaluate its ability to protect against sea lice infestations. A significant reduction in the number of parasites per fish was observed at 24 days post-challenge. These results, together with the delay observed in the development of parasites from the vaccinated group suggest that the major effect of immunization was on the second parasite generation. The results of these experiments suggest that the my32 protein may be a promising target for vaccine development to control sea lice infestations in fish.


Assuntos
Copépodes/genética , Copépodes/parasitologia , Ectoparasitoses/veterinária , Doenças dos Peixes/prevenção & controle , Doenças Parasitárias em Animais/prevenção & controle , Vacinação/métodos , Animais , Copépodes/imunologia , Ectoparasitoses/imunologia , Ectoparasitoses/prevenção & controle , Doenças dos Peixes/imunologia , Inativação Gênica , Dados de Sequência Molecular , Doenças Parasitárias em Animais/imunologia , Análise de Sequência de DNA , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia
20.
J Biotechnol ; 151(2): 175-9, 2011 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-21112358

RESUMO

The importance of somatotropin as a growth promoting agent and immune-stimulator has long been recognized and its potential application in the fish farming industry has been an active research area. In the work reported here, we sought to improve the stability of a previously obtained truncated somatotropin by applying a 60 °C heat shock to the culture supernatant containing this molecule, and then compared its effects with and without heat shock on larval growth and immune functions. We observed that the treatment with heat shock at 60 °C enhanced protein stability, growth and innate immune functions in tilapia larvae.


Assuntos
Hormônio do Crescimento/fisiologia , Pichia/metabolismo , Animais , Western Blotting , Meios de Cultura/metabolismo , Hormônio do Crescimento/química , Hormônio do Crescimento/metabolismo , Proteínas de Choque Térmico/química , Resposta ao Choque Térmico , Temperatura Alta , Lectinas , Espécies Reativas de Oxigênio , Temperatura , Tilápia , Fatores de Tempo
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