A recombinant chimera composed of repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evaluation of specific IgG response in mice and pigs (2023)

Table of Contents

Veterinary Microbiology Abstract Introduction Section snippets Reagents Results Discussion Acknowledgements References (26) Immunogenicity of Actinobacillus ApxIA toxin epitopes fused to the E. coli heat-labile enterotoxin B subunit Vaccine Identification of a novel adhesin-like glycoprotein from Mycoplasma hyopneumoniae Vet. Microbiol. V3-loop Pseudomonas exotoxin chimeras: characterization of novel candidates for vaccination against human immunodeficiency virus-1 J. Biol. Chem. Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems Vaccine Structure and function relationship of Pseudomonas exotoxin A: an immunochemical study J. Biol. Chem. Functional domain of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli Cell Mucosal administration of a chimera composed of Pseudomonas exotoxin and the gp120 V3 loop sequence of HIV-1 induces both salivary and serum antibody responses Vaccine Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin J. Mol. Biol. The human 2-macroglobulin receptor: identification of a 420 kD cell surface glycoprotein specific for the activated conformation of 2-macroglobulin J. Cell Biol. Membrane receptors for bacterial toxins Microbiol. Rev. Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare: a survey Nord. Vet. Med. Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin Infect. Immun. Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae J. Bacteriol. Cited by (26) A concise review of vaccines against Mycoplasma hyopneumoniae Analysis of swine antigen-specific antibody responses to Mycoplasma hyopneumoniae infection determined by protein microarray Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs Mycoplasma hyopneumoniae: From disease to vaccine development Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins Production and characterization of recombinant transmembrane proteins from Mycoplasma hyopneumoniae Recommended articles (6) Specific Character of Anaerobic Bacterial Infections in Patients Treated in Transplantation Wards at One of the Clinical Hospitals in Warsaw Antibodies to VP1 of swine pasivirus in humans without evidence of transmission from a pig source NHERF2 Protein Mobility Rate Is Determined by a Unique C-terminal Domain That Is Also Necessary for Its Regulation of NHE3 Protein in OK Cells The diversity of the immune response to the A2 domain of human factor VIII Clinical and laboratory-derived parameters of 119 hospitalized patients with coronavirus disease 2019 in Xiangyang, Hubei Province, China Targeting antigens to Dec-205 on dendritic cells induces a higher immune response in chickens: Hemagglutinin of avian influenza virus example

Veterinary Microbiology

Volume 80, Issue 4,

22 June 2001

, Pages 347-357

Author links open overlay panel, , ,

https://doi.org/10.1016/S0378-1135(01)00315-7 Get rights and content

Abstract

Using the binding and translocation domain of Pseudomonas exotoxin A [domain III deleted PE termed PE(ΔIII)] as a vehicle, this study characterized and evaluated a novel application of PE toxin in Mycoplasma hyopneumoniae adhesin used as an immunogen. PCR and sequence analysis revealed that 16 copies of AAKPV(E) in tandem repeat region 1 (RR1) of M. hyopneumoniae 97kDa adhesion were successfully fused to the downstream of PE(ΔIII) to create a subunit vaccine, i.e. PE(ΔIII)-RR1. This chimeric protein, over-expressed in inclusion bodies of E. coli BL21(DE3)pLysS, was characterized by a monoclonal antibody (MAb) F2G5 prepared against RR1 of the 97kDa adhesin and was readily purified. The data indicated that the epitope recognized by MAb F2G5 was located in the structure of PE(ΔIII)-RR1. Using ELISA and Western blot analyses, the specific IgG immune response against RR1 and whole adhesin in mice immunized with PE(ΔIII)-RR1 was found more marked than that in mice immunized with the M. hyopneumoniae whole cells. Similarly, PE(ΔIII)-RR1 also stimulated a remarkable IgG response against RR1 in pigs compared to that in pigs immunized with the conventional M. hyopneumoniae vaccine. The PE(ΔIII)-RR1 would be potentially useful for the future development of a M. hyopneumoniae adhesin vaccine.

Introduction

Mycoplasma hyopneumoniae of the class Mollicutes (meaning a cell wall-less bacterium), is a globally and economically important pathogen that causes mycoplasmal pneumonia of swine (MPS) (Ross, 1986). Vaccination appears to be the more effective means of either eradicating or controlling the infection than antibiotics (Kavanagh, 1994, Kobisch and Friis, 1996). The most commonly used vaccines were prepared from killed M. hyopneumoniae whole cells (Kobisch and Friis, 1996, Le Grand and Kobisch, 1996). Nevertheless, mass production of M. hyopneumoniae vaccine employing the complicated cultivation method is costly and time consuming. Following the discovery of bacterial adhesins, the development of a bacterial adhesin vaccine through recombinant techniques has recently been considered (O’Hagan, 1998, Keil et al., 1999, Novotny et al., 2000). Zhang et al. (1995) recently identified a M. hyopneumoniae 97kDa molecule functionally correlated with adhesin using a monoclonal antibody (MAb)F2G5. Hsu et al., 1997, Hsu and Minion, 1998 subsequently identified the cilia binding site as the proline-rich AAKPV(E) repeat region 1 (RR1) located at the carboxy terminus in cloned P97 gene and Williamson (1994) found that the RR1 protein is highly immunogenic. Since RR1 is the potential candidate to develop the M. hyopneumoniae adhesin vaccine, we cloned the RR1 gene from Mycoplasma hyopneumoniae Fl04-4 and then constructed this gene in the pET protein expression system in a preliminary experiment. However, the amount of RR1 protein expressed in E. coli BL21(DE3)pLysS was relatively low and further complicated the purification processes and limited its application in subunit vaccine. Recently, this problem was resolved by the utilization of domain III deleted Pseudomonas exotoxin A constructed in pET expression vector as a mass production system [pPE(ΔIII)] via the formation of inclusion bodies of PE(ΔIII) and the fused foreign proteins (Hwang et al., 1987).

Pseudomonas exotoxin A (PE) consists of three domains in the 613 amino acid residues. Domain I is for receptor binding, domain II, for membrane translocation promoting access to the cytosol, and domain III is responsible for the catalytic inactivation of protein synthesis via the ADP-ribosylation of elongation factor 2 and results in cell death (Eidels et al., 1983, Middlebrook and Dorland, 1984, Taupiac et al., 1999). The PE(ΔIII) can be used to deliver a foreign protein into cells through receptor mediated endocytosis (Hwang et al., 1987, Hwang and Chen, 1989 and function as the non-toxic bioadjuvant, such as cholera (CT) or E. coli enterotoxins (LT) do, thereby enhancing the immune response to genetically fused antigens (Holmgren et al., 1993, Sixma et al., 1993, Bagdasarian et al., 1999). This application was recently used by Mrsny et al. (1999) to carry the gp 120v3 loop sequence of HIV-1, successfully inducing both systemic and mucosal responses.

In this study, we combined the RR1 gene with PE(ΔIII) gene and over-expressed it in the inclusion body. The immunogenicity of RR1 in the PE(ΔIII)-RR1 fusion protein was then determined in both mice and pigs.

Section snippets

Reagents

The nontoxic form of Pseudomonas exotoxin devoid of domain III [PE(ΔIII)] was kindly donated from Dr. Hwang et al. (1987). Monoclonal antibody (MAb) F2G5 recognizing the RR1 fragment of P97 adhesin was obtained from Dr. Ross (Iowa State University, Ames, IA, USA) and has been previously characterized (Zhang et al., 1995). Goat anti-mouse IgG and anti-pig IgG conjugated with alkaline phosphatase were purchased from Chemicon International Inc. (USA) and Jackson Laboratories Inc. (USA),

Results

Fig. 1 shows the strategy to construct PE(ΔIII)-RR1. pPE(ΔIII) was generated from domain III deleted full length PE toxin gene and then the RR1 gene was inserted into the down stream of PE(ΔIII) at the Aat II and EcoR I sites.

The RR1 from PCR product was sequenced and found to contain 16 copies of the 5-amino acid tandem repeats (unpublished). Under the control of the T7 promoter-driven system, although the 16kDa RR1 was expressed, the amount was relatively low. Using the same control system of

Discussion

Although development of an adhesin vaccine of M. hyopneumoniae needs to be fully explored, the amount of adhesin isolated from Mycoplasma via conventionally prepared culture-medium is limited and the purification procedures for whole adhesin molecule are relatively complex and tedious (Zhang et al., 1995, Chen et al., 1998). However, in this study, this problem was overcome by using the recombinant technique via cloning and expressing the cilia binding fragment (RR1) containing 102 amino acids

Acknowledgements

The authors would like to thank the Council of Agriculture of the Republic of China for financially supporting this research under Contract No. 89-BT-2.3-BQ-61(11).

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See Also
Human Recombinant A2M/CPAMD5/Alpha-2-macroglobulin Protein (His Tag) (from Baculovirus Insect cells)The electrophoretically 'slow' and 'fast' forms of the alpha 2-macroglobulin molecule. - PDF Download Free B2MU - Overview: Beta-2 Microglobulin, Random, Urine Antioksidativno ponašanje a2-makroglobulina kod degeneracije intervertebralnog diska
J. Biol. Chem.
(1989)
J. Hwang et al.
Functional domain of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli
Cell
(1987)
R.J. Mrsny et al.
Mucosal administration of a chimera composed of Pseudomonas exotoxin and the gp120 V3 loop sequence of HIV-1 induces both salivary and serum antibody responses
Vaccine
(1999)
T.K. Sixma et al.
Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin
J. Mol. Biol.
(1993)
J.D. Ashcom et al.
The human 2-macroglobulin receptor: identification of a 420 kD cell surface glycoprotein specific for the activated conformation of 2-macroglobulin
J. Cell Biol.
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L. Eidels et al.
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Microbiol. Rev.
(1983)

N.F. Friis

Some recommendations concerning primary isolation of Mycoplasma suipneumoniae and Mycoplasma flocculare: a survey

Nord. Vet. Med.

(1975)

T. Hsu et al.

Identification of the cilium binding epitope of the Mycoplasma hyopneumoniae P97 adhesin

Infect. Immun.

(1998)

T. Hsu et al.

Cloning and functional analysis of the P97 swine cilium adhesin gene of Mycoplasma hyopneumoniae

J. Bacteriol.

(1997)

Cited by (26)

A concise review of vaccines against Mycoplasma hyopneumoniae
2019, Research in Veterinary Science
Citation Excerpt :
King et al. expressed that vaccination with Mhp1, a protein of Mhp similar to P97, offers minimal protection in pigs (King et al., 1997). To enhance this protective immune effect, Chen et al. found that a chimeric protein consisting of P97R1 and Pseudomonas aeroginosa exotoxin A could induce a specific immunoglobulin G (IgG) immune response in mice and pigs (Chen et al., 2001). Shimoji et al. reported that pigs intranasally (IN) immunized with an attenuated Erysipelothrix rhusiopathiae YS-19 strain expressing the C-terminal portion of P97 had significantly fewer lung lesions, but specific serum antibodies were not observed (Shimoji et al., 2003).
Mycoplasma hyopneumoniae (Mhp) is the pathogen of Mycoplasmal pneumonia of swine (MPS), a chronic respiratory infectious discease which causes enormous losses to the swine industry worldwide. At present, vaccination is the most effective mean to prevent and reduce economic losses caused by this pathogen. Currently, MPS vaccines mainly include inactivated vaccines and attenuated live vaccines. However, these approved vaccines still have many drawbacks, and the infection of Mhp has not yet been fully elucidated. Adhesion factors of Mhp have been shown to play a direct role in the pathogen's adherence, and thus were given consideration to be included in the composition of the vaccine. This shows a good prospect due to the advantages and feasibility of genetically engineering a vaccine. In this review, we summarize the work of researchers in recent years about the development of vaccines against Mhp, and we focus on the development of genetically engineering vaccines and some novel combined vaccines.
Analysis of swine antigen-specific antibody responses to Mycoplasma hyopneumoniae infection determined by protein microarray
2019, Veterinary Microbiology
Citation Excerpt :
Shimoji et al. showed a distinct lack of antibody response against P97 when an Erysipelothrix rhusiopathiae strain expressing the C-terminal portion of P97 was used as a vaccine in pigs (Shimoji et al., 2002). Antibody responses against P97 R1 could be generated if a R1-containing subunit peptide was adjuvanted (Conceicao et al., 2006; Chen et al., 2001), but normal whole cell vaccines failed to induce significant antibodies against P97 (Fisch et al., 2016). In contrast, Okamba et al. demonstrated partial protection using an adenovirus expressing the C-terminal portion of P97 (Okamba et al., 2010).
Pigs harbor several different species of mycoplasmas, of which Mycoplasma hyopneumoniae presents the most significant economic impact on the swine industry. While ELISAs are the predominant diagnostic assay to measure antibody responses during infection with M. hyopneumoniae, the assay itself is only a rough estimate of the total antibody response. It lends little information on pathogen-wide antigen-specific responses. In addition, antibody responses to M. hyopneumoniae as measured by ELISA are slow to develop in infected swine. Our goal was to determine if a protein microarray could be more sensitive and informative of the serological responses of pigs to M. hyopneumoniae infection. The gene sequences of approximately 50 M. hyopneumoniae surface proteins or protein fragments were cloned, mutated to remove UGA codons, expressed in Escherichia coli and purified. The arrays were used to interrogate pig sera from various sources. Sera from naturally-infected swine gave some variability in antigen-specific responses, but, unexpectedly, the responses against the C-terminal portion of the major adhesin P97 was weak in all animals, including those that were experimentally infected. In two of four 118-day experimentally-infected caesarian-derived colostrum-deprived pigs, the strongest antibody responses occurred on days 30 and 54 against members of the P97/P102 paralog families. Our Day 0 results in the other two animals indicate that although thought to be mycoplasma free by all known criteria (serology and PCR), they may have harbored an inapparent Mycoplasma infection. In summary, the protein microarray has the potential to identify new targets for assay development to enhance sensitivity of antibody-based assays.
Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs
2014, Vaccine
Citation Excerpt :
Extensive characterisation of the surface-accessible Mhp P97/P102 paralog family members has found them to be multifunctional cilium adhesins which exploit host surface glycoconjugates and extracellular matrix components to facilitate Mhp pathogenesis [3–13]. Despite the broad range of host molecule interactions displayed by the P97/P102 paralog family of proteins, only P97 has been investigated as a potential vaccine candidate [14–16]. Following colonisation of cilia, Mhp induces the secretion of numerous cytokines including interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α [17–21].
Pig responses to recombinant subunit vaccines containing fragments of eight multifunctional adhesins of the Mycoplasma hyopneumoniae (Mhp) P97/P102 paralog family formulated with Alhydrogel^® or Montanide™ Gel01 were compared with a commercial bacterin following experimental challenge. Pigs, vaccinated intramuscularly at 9, 12 and 15 weeks of age with either of the recombinant formulations (n=10 per group) or Suvaxyn^® M. hyo (n=12), were challenged with Mhp strain Hillcrest at 17 weeks of age. Unvaccinated, challenged pigs (n=12) served as a control group. Coughing was assessed daily. Antigen-specific antibody responses were monitored by ELISA in serum and tracheobronchial lavage fluid (TBLF), while TBLF was also assayed for cytokine responses (ELISA) and bacterial load (qPCR). At slaughter, gross and histopathology of lungs were quantified and damage to epithelial cilia in the porcine trachea was evaluated by scanning electron microscopy. Suvaxyn^® M. hyo administration induced significant serological responses against Mhp strain 232 whole cell lysates (wcl) and recombinant antigen F3_P216, but not against the remaining vaccine subunit antigens. Alhydrogel^® and Montanide™ Gel01-adjuvanted antigen induced significant antigen-specific IgG responses, with the latter adjuvant eliciting comparable Mhp strain 232 wcl specific IgG responses to Suvaxyn^® M. hyo. No significant post-vaccination antigen-specific mucosal responses were detected with the recombinant vaccinates. Suvaxyn^® M. hyo was superior in reducing clinical signs, lung lesion severity and bacterial load but the recombinant formulations offered comparable protection against cilial damage. Lower IL-1β, TNF-α and IL-6 responses after challenge were associated with reduced lung lesion severity in Suvaxyn^® M. hyo vaccinates, while elevated pathology scores in recombinant vaccinates corresponded to cytokine levels that were similarly elevated as in unvaccinated pigs. This study highlights the need for continued research into protective antigens and vaccination strategies that will prevent Mhp colonisation and establishment of infection.
Mycoplasma hyopneumoniae: From disease to vaccine development
2013, Veterinary Microbiology
Citation Excerpt :
King et al. (1997) reported that a subunit vaccine based on recombinant adhesin P97 did not induce significant protection in challenged pigs. However, when the C-terminal region of P97 (R1) was fused to Pseudomonas toxin A, immunized mice and pigs produced specific immune responses against R1 (Chen et al., 2001). Oral vaccination with recombinant E. rhusiopathiae strains expressing the P97 protein reduced the severity of pneumonic lung lesions caused by M. hyopneumoniae infection, showing that E. rhusiopathiae may be a promising vaccine vector for delivering foreign antigens to the immune systems of pigs (Ogawa et al., 2009).
Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), a disease that affects swine production worldwide. Vaccination is the most cost-effective strategy for the control and prevention of the disease. Despite efforts to control M. hyopneumoniae infection, significant economic losses in pig production continue to occur. The results of genome-based research have the potential to help understand the biology and pathogenesis of M. hyopneumoniae, and contribute to the development of more effective vaccines and diagnostic tests. In this review, the characteristics of M. hyopneumoniae related to pathogenesis and control measures will be discussed. Special emphasis will be placed on vaccination strategies that have been proposed with the use of reverse vaccinology approaches.
Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins
2012, Comparative Immunology, Microbiology and Infectious Diseases
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However, these vaccines are not enough to eliminate M. hyopneumoniae from infected pig herds [4]. Investigation of new vaccines is actively occurring, especially recombinant subunit and naked DNA vaccines [4–15]. These strategies have the potential to result in a new vaccine able to control M. hyopneumoniae infection.
Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.
Production and characterization of recombinant transmembrane proteins from Mycoplasma hyopneumoniae
2012, Veterinary Microbiology
Citation Excerpt :
However, vaccination with the currently available vaccines is not able to prevent transmission or establishment of the microorganism in the lungs (Thacker et al., 1998; Meyns et al., 2006). These vaccines consist of inactivated whole-cell preparations (bacterin), and the vaccine is expensive (Chen et al., 2001), as in vitro growth of M. hyopneumoniae requires a rich medium and is a time consuming process (Thacker, 2006). Besides, more efficient and cheaper vaccines and improved diagnostic assays could contribute for the control of EP.
Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia (EP), a chronic respiratory disease which causes significant economic losses to the swine industry worldwide. More efficient strategies for controlling this disease are necessary. In this study, we cloned17 genes coding for transmembrane proteins from M. hyopneumoniae, among which six were successfully expressed in Escherichia coli and had their immunogenic and antigenic properties evaluated. All proteins were immunogenic in mice and sera from naturally infected pigs reacted with the recombinant proteins, suggesting that they are expressed during infection. These antigens may contribute for the development of new recombinant vaccines and diagnostic tests against EP.

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