PCV Cap proteins fused with calreticulin expressed into polymers in Escherichia coli with high immunogenicity in mice

Abstract Background：Porcine circovirus type 2 (PCV2) is considered as one of the critical viral pathogens of porcine circovirus diseases (PCVDs), which results in economic losses in the pig breeding industry. Capsid protein (Cap) of PCV2 acts as the most protective antigen in the course of immune response. Therefore, developing a novel and safe subunit vaccine against PCV2 infection is needed.Results: In this study, the Cap gene was bound to the truncated calreticulin (CRT) (120-250 aa/120-308 aa) at the N/C terminal, and then the CRT-Cap fusion genes were expressed in Escherichia coli (E.coli). The size-exclusion chromatography and dynamic light scattering (DLS) data showed that the purified recombinant CRT-Cap fusion protein (rP5F) existed in the form of polymers. The results of ELISA and NA indicated that humoral immune responses induced by rP5F in mice were almost identical to those by the commercial subunit and inactivated vaccines. The lymphocyte proliferation and cytokine secretion were also detected in rP5F immunized mice. According to the results of PCV2-challenge experiment, the virus loads significantly decreased in mice immunized with rP5F. The data obtained in the current study revealed that rP5F had the potential to be a subunit vaccine candidate against PCV2 in the future.Conclusions: We have successfully expressed Cap-CRT fusion proteins in E.coli and optimized rP5F could form into immunogenic polymers. Mice immunized with rP5F efficiently induced humoral and part of cellular immune responses and decreased the virus content against PCV2-challenge, which suggested that rF5P could be a potential subunit vaccine candidate.

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PCV Cap proteins fused with calreticulin expressed into polymers in Escherichia coli with high immunogenicity in mice

10.21203/rs.3.rs-15890/v1 ◽

2020 ◽

Author(s):

Chang Liu ◽

Yunchao Liu ◽

Hua Feng ◽

Baolei Zhao ◽

Yumei Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Immune Responses ◽

Subunit Vaccine ◽

Protective Antigen ◽

Vaccine Candidate ◽

Porcine Circovirus Type ◽

Size Exclusion ◽

Porcine Circovirus ◽

Economic Losses ◽

Virus Content

Abstract Background：Porcine circovirus type 2 (PCV2) is considered one of the critical viral pathogens of porcine circovirus diseases (PCVDs), which results in economic losses in the pig breeding industry. Capsid protein (Cap) of PCV2 acts as the most protective antigen in the course of immune response. Therefore, developing a novel and safe subunit vaccine against PCV2 infection is needed.Results: In this study, the Cap gene bound to the truncated calreticulin (CRT) (120-250 aa/120-308 aa) at the N/C terminal, and then the CRT-Cap fusion genes were expressed in Escherichia coli (E.coli). The size-exclusion chromatography and dynamic light scattering (DLS) data showed that the purified recombinant CRT-Cap fusion protein (rP5F) existed in the form of polymers. The results of ELISA and NA indicated that humoral immune responses induced by rP5F in mice were almost identical to those by the commercial subunit and inactivated vaccine. The lymphocyte proliferation and cytokine secretion were also detected in rP5F immunized mice. According to the results of the PCV2-challenge experiment, virus loads significantly decreased in mice immunized with rP5F. The data obtained in the current study revealed that rP5F might have the potential to be a subunit vaccine candidate against PCV2 in the future.Conclusions: We have successfully expressed Cap-CRT fusion proteins in E.coli and optimized rP5F could form into immunogenic polymers. Mice immunized with rP5F efficiently induced humoral and part of cellular immune responses and decreased the virus content against PCV2-challenge, which suggested that rF5P could be a potential subunit vaccine candidate.

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Swine Influenza Virus Infection Decreases the Protective Immune Responses of Subunit Vaccine Against Porcine Circovirus Type 2

Frontiers in Microbiology ◽

10.3389/fmicb.2021.807458 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuhang Sun ◽

Jinlong Zhang ◽

Zixuan Liu ◽

Ying Zhang ◽

Kehe Huang

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Subunit Vaccine ◽

Swine Influenza Virus ◽

Swine Influenza ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Siv Infection

Porcine circovirus type 2 (PCV2) is the primary pathogen of porcine circovirus diseases and porcine circovirus associated diseases. Immunization with a vaccine is considered an effective measure to control these diseases. However, it is still unknown whether PCV2 vaccines have protective immune responses on the animals infected with swine influenza virus (SIV), a pandemic virus in swine herds. In this study, we first compared the effects of 2 different PCV2 vaccines on normal mice and SIV-infected mice, respectively. The results showed that these two vaccines had protective immune responses in normal mice, and the subunit vaccine (vaccine S) had better effects. However, the inactivated vaccine (vaccine I) instead of vaccine S exhibited more immune responses in the SIV-infected mice. SIV infection significantly decreased the immune responses of vaccine S in varying aspects including decreased PCV2 antibody levels and increased PCV2 replication. Mechanistically, further studies showed that SIV infection increased IL-10 expression and M2 macrophage percentage, but decreased TNF-α expression and M1 macrophage percentage in the mice immunized with vaccine S; on the contrary, macrophage depleting by using clodronate-containing liposomes significantly alleviated the SIV infection-induced decrease in the protective immune responses of vaccine S against PCV2. This study indicates that SIV infection decreases the protective immune responses of vaccine S against PCV2. The macrophage polarization induced by SIV infection might facilitate decreased immune responses to vaccine S, which provides new insight into vaccine evaluation and a reference for the analysis of immunization failure.

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A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate

Viruses ◽

10.3390/v12040385 ◽

2020 ◽

Vol 12 (4) ◽

pp. 385

Author(s):

Emilio Lamazares ◽

Fernando Gutiérrez ◽

Angela Hidalgo ◽

Nicolas A. Gutiérrez ◽

Felipe I. Espinoza ◽

...

Keyword(s):

Vaccine Development ◽

Vaccine Candidate ◽

Low Cost ◽

Chimeric Protein ◽

Structural Complexity ◽

Porcine Circovirus Type ◽

Porcine Circovirus ◽

Economic Losses ◽

Tobacco Necrosis Virus ◽

Effective Vaccine

Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.

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Maternally Derived Antibody Levels Influence on Vaccine Protection against PCV2d Challenge

Animals ◽

10.3390/ani11082231 ◽

2021 ◽

Vol 11 (8) ◽

pp. 2231

Author(s):

István Kiss ◽

Krisztina Szigeti ◽

Zalán G. Homonnay ◽

Vivien Tamás ◽

Han Smits ◽

...

Keyword(s):

Immune Response ◽

Humoral Immune Response ◽

Subunit Vaccine ◽

Porcine Circovirus Type ◽

Porcine Circovirus ◽

Vaccine Protection ◽

Humoral Immune ◽

Negative Effect ◽

Antibody Levels

Piglets from a porcine circovirus type 2 (PCV2) stable farm of low and high levels of maternally derived antibodies (MDA) against PCV2 were vaccinated either with a whole virus type or a PCV2 ORF2 antigen-based commercial subunit vaccine at three weeks of age. Two non-vaccinated groups served as low and high MDA positive controls. At four weeks post vaccination, all piglets were challenged with a PCV2d-2 type virus strain and were checked for parameters related to vaccine protection over a four-week observation period. MDA levels evidently impacted the outcome of the PCV2d-2 challenge in non-vaccinated animals, while it did not have a significant effect on vaccine-induced protection levels. The humoral immune response developed faster in the whole virus vaccinates than in the subunit vaccinated pigs in the low MDA groups. Further, high MDA levels elicited a stronger negative effect on the vaccine-induced humoral immune response for the subunit vaccine than for the whole virus vaccine. The group-based oral fluid samples and the group mean viraemia and faecal shedding data correlated well, enabling this simple, and animal welfare-friendly sampling method for the evaluation of the PCV2 viral load status of these nursery piglets.

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Immunogenicity and protective efficacy of enterotoxigenic Escherichia coli (ETEC) total RNA against ETEC challenge in a mouse model

Scientific Reports ◽

10.1038/s41598-020-77551-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Mandi Liu ◽

Yue Zhang ◽

Di Zhang ◽

Yun Bai ◽

Guomei Liu ◽

...

Keyword(s):

Escherichia Coli ◽

Mouse Model ◽

Immune Responses ◽

Protective Efficacy ◽

Enterotoxigenic Escherichia Coli ◽

Economic Losses ◽

Th2 Response ◽

Total Rna ◽

Etec Infection

AbstractEnterotoxigenic Escherichia coli (ETEC), an essential cause of post-weaning diarrhea (PWD) in piglets, leads to significant economic losses to the pig industry. The present study aims to identify the role of ETEC total RNA in eliciting immune responses to protect animals against ETEC infection. The results showed that the total RNA isolated from pig-derived ETEC K88ac strain effectively stimulated the IL-1β secretion of porcine intestinal epithelial cells (IPEC-J2). The mouse model immunized with ETEC total RNA via intramuscular injection (IM) or oral route (OR) was used to evaluate the protective efficiency of the ETEC total RNA. The results suggested that 70 μg ETEC total RNA administered by either route significantly promoted the production of the serum IL-1β and K88ac specific immunoglobulins (IgG, IgM, and IgA). Besides, the ETEC RNA administration augmented strong mucosal immunity by elevating K88ac specific IgA level in the intestinal fluid. Intramuscularly administered RNA induced a Th1/Th2 shift toward a Th2 response, while the orally administered RNA did not. The ETEC total RNA efficiently protected the animals against the ETEC challenge either by itself or as an adjuvant. The histology characterization of the small intestines also suggested the ETEC RNA administration protected the small intestinal structure against the ETEC infection. Particularly of note was that the immunity level and protective efficacy caused by ETEC RNA were dose-dependent. These findings will help understand the role of bacterial RNA in eliciting immune responses, and benefit the development of RNA-based vaccines or adjuvants.

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Immune Responses of Piglets Immunized by a Recombinant Plasmid Containing Porcine Circovirus Type 2 and Porcine Interleukin-18 Genes

Viral Immunology ◽

10.1089/vim.2014.0036 ◽

2014 ◽

Vol 27 (10) ◽

pp. 521-528 ◽

Cited By ~ 3

Author(s):

Guang-Lei Chen ◽

Peng-Fei Fu ◽

Lin-Qing Wang ◽

Xin-Sheng Li ◽

Hong-Ying Chen

Keyword(s):

Immune Responses ◽

Recombinant Plasmid ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus ◽

Interleukin 18

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Histopathological and virological findings in emaciated pigs from Mexico: an exploratory study

Acta Veterinaria Brno ◽

10.2754/avb201887030213 ◽

2018 ◽

Vol 87 (3) ◽

pp. 213-217

Author(s):

Aide Alpízar ◽

Joaquim Segalés ◽

Simón Martínez ◽

Atalo Martínez ◽

Guadalupe Socci ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Body Condition ◽

Porcine Circovirus Type ◽

Porcine Circovirus ◽

Economic Losses ◽

Respiratory Syndrome Virus ◽

Chain Reaction ◽

Polymerase Chain ◽

Histopathological Studies ◽

Weaning Piglets

The objective of this work was to detect the presence of three main pig respiratory viral agents (porcine rubulavirus [PorPV], porcine circovirus type 2 [PCV-2], and porcine reproductive and respiratory syndrome virus [PRRSV]) in tissues of emaciated piglets from the Baj'o Region (Mexico). Necropsies and histopathological studies of 37 pigs with poor body condition were performed; viruses were detected by molecular biology methods and PCV-2 was further assessed by immunohistochemistry (IHC). Histopathologically, interstitial pneumonia was observed in 25/37 (68%) of the piglets. Also, a varying degree of lymphocyte depletion in lymphoid organs was found in 14/37 (38%) animals. Through polymerase chain reaction (PCR) and/or reverse transcription polymerase chain reaction (RT-PCR), from the 37 pigs, 16 were positive for PCV-2, 18 for PRRSV and 1 for PorPV. In accordance with these results, the infection and/or co-infection with PCV-2 and PRRSV were fairly frequent findings in piglets with poor body condition in Mexico, while the infection by PorPV was apparently negligible. Wasting of post-weaning piglets is a global pig farming problem that causes great economic losses and has been associated with diverse factors: microbial agents, environmental factors, nutritional factors, and management. When the Blue Eye Disease was first reported in Mexico, it was associated with severe wasting in post-weaning piglets. This study demonstrated that this disease does not seem to play such an important role in the wasting as was previously thought.

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Comparing the efficiency of different Escherichia coli strains in producing recombinant capsid protein of porcine circovirus type 2

Molecular and Cellular Probes ◽

10.1016/j.mcp.2020.101556 ◽

2020 ◽

Vol 52 ◽

pp. 101556 ◽

Cited By ~ 1

Author(s):

Vishal Rai ◽

Vikramaditya Upmanyu ◽

Gulam Mohd ◽

Ravi Kumar ◽

Sanganagouda Koppad ◽

...

Keyword(s):

Escherichia Coli ◽

Capsid Protein ◽

Porcine Circovirus Type ◽

Porcine Circovirus Type 2 ◽

Porcine Circovirus

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Reduction in Oviposition of Poultry Red Mite (Dermanyssus gallinae) in Hens Vaccinated with Recombinant Akirin

Vaccines ◽

10.3390/vaccines7030121 ◽

2019 ◽

Vol 7 (3) ◽

pp. 121 ◽

Cited By ~ 6

Author(s):

Jose Lima-Barbero ◽

Marinela Contreras ◽

Kathryn Bartley ◽

Daniel Price ◽

Francesca Nunn ◽

...

Keyword(s):

Egg Production ◽

Protective Antigen ◽

Vaccine Candidate ◽

Protective Efficacy ◽

Egg Yolk ◽

Economic Losses ◽

Dermanyssus Gallinae ◽

Poultry Red Mite ◽

Production Sector ◽

Worldwide Distribution

The poultry red mite (PRM), Dermanyssus gallinae, is a hematophagous ectoparasite of birds with worldwide distribution that causes economic losses in the egg-production sector of the poultry industry. Traditional control methods, mainly based on acaricides, have been only partially successful, and new vaccine-based interventions are required for the control of PRM. Vaccination with insect Akirin (AKR) and its homolog in ticks, Subolesin (SUB), have shown protective efficacy for the control of ectoparasite infestations and pathogen infection/transmission. The aim of this study was the identification of the akr gene from D. gallinae (Deg-akr), the production of the recombinant Deg-AKR protein, and evaluation of its efficacy as a vaccine candidate for the control of PRM. The anti-Deg-AKR serum IgY antibodies in hen sera and egg yolk were higher in vaccinated than control animals throughout the experiment. The results demonstrated the efficacy of the vaccination with Deg-AKR for the control of PRM by reducing mite oviposition by 42% following feeding on vaccinated hens. A negative correlation between the levels of serum anti-Deg-AKR IgY and mite oviposition was obtained. These results support Deg-AKR as a candidate protective antigen for the control of PRM population growth.

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