Pasteurella multocida PlpE Protein Polytope as a Potential Subunit Vaccine Candidate

Abstract

Pasteurella multocida is the causative agent of a range of animal, and occasionally human, diseases. Problems with antimicrobial treatment of P. multocida highlight the need to find other possible ways, such as prophylaxis, to manage infections. Current vaccines against P. multocida include inactivated bacteria, live attenuated and nonpathogenic bacteria; they have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence. Using bioinformatics approaches, potentially immunogenic and protective epitopes were identified and merged to design the most optimally immunogenic triple epitope PlpE fusion protein of P. multocida as a vaccine candidate. This triple epitope (PlpE1 + 2 + 3) was cloned into the pBAD/gIII A plasmid (pBR322-derived expression vectors designed for regulated, secreted recombinant protein expression and purification in Escherichia coli), expressed in Top 10 E. coli and purified in denatured form using Ni-NTA chromatography and 8 M urea. The immunogenicity of the purified proteins in BALB/c mice was assayed by measuring immunoglobulin G (IgG) responses. The protection potential was evaluated by challenging with 10 LD50 of serotype A:1, X-73 strain of P. multocida and compared with commercially available inactivated fowl cholera vaccine and PlpE protein. IgG levels elicited by the polytope fusion protein of P. multocida PlpE were higher than both commercially available inactivated fowl cholera vaccine and PlpE protein. Surprisingly, protection was independent of IgG level; commercially available inactivated fowl cholera vaccine (100% protection) was more protective than the polytope fusion protein (69% protection) and PlpE protein (69% protection). These results also confirm that IgG level is not a reliable indicator of protection. Further studies to evaluate the other antibody classes, such as immunoglobulin A or M, are required. The role of cell-mediated immunity should also be considered as a potential protection pathway.

Impacts

PlpE protein polytopes of Pasteurella multocida is a good subunit vaccine candidate against P. multocida infection.

PlpE protein polytopes of P. multocida would be successful to raise the immune response against all P. multocida serotypes.

PlpE protein polytopes of P. multocida has equal protection as PlpE protein against P. multocida infection.

Introduction

P asteurella multocida is a Gram-negative, nonmotile, nonspore-forming, and facultative aerobic/anaerobic coccobacillus, which is the etiological agent of a wide range of animal diseases, including pneumonia in cattle and sheep, atrophic rhinitis in swine, hemorrhagic septicemia in buffalo and cattle, and fowl cholera in poultry (Peng et al., 2019). P. multocida-related infections thus cause economic loss in sheep, goats, rabbits, poultry, and other livestock industries. P. multocida infections have high mortality rates, but also cause production losses due to reductions in weight gain (Wilson and Ho, 2013). This bacterium is responsible for 30% of total cattle deaths around the world and losses of one billion dollars annually in this industry in North America alone (Roier et al., 2013). P. multocida has also been isolated from healthy animals as a member of the oropharyngeal flora of calves, where it may not cause serious disease, but in stress conditions, it is one of the bacterial pathogens associated with disease (Ujvári et al., 2019). P. multocida strains are classified into five capsular serogroups (A, B, D, E, and F) and 16 somatic lipopolysaccharide serotypes numbered from 1 to 16 (Farahani et al., 2019).

Vaccination of livestock plays a vital role in improving animal health and welfare and preventing animal to human transmission, which is one of the main goals of public health strategies. Present vaccine candidates against this agent include live attenuated vaccines, which have several disadvantages such as induction of short-term or ineffective immunity (Hofacre and Glisson, 1986, Luo et al., 2005) and also possibility of reversion to virulence in cases where the attenuation mechanism is not defined (Hopkins et al., 2016). Killed vaccines are not ideal because of variable efficacy. They generally stimulate short-term immunity and the vaccinated animals are protected only against the homologous serotype (Mariana and Hirst, 2000, Ahmad et al., 2014).

Considering the limitations of current vaccines, there is a clear need to develop vaccines that have no risk in causing disease, have minimal side effects, can overcome any antigenic variation, and are stable and easy to prepare. One such approach is the development of new subunit vaccines, which would have advantages such as easier production and avoid the possibility of reversion to virulence (Hansson et al., 2000).

Hatfaludi et al. (2012) assessed 71 candidate proteins identified by reverse vaccinology. This study concluded that the conserved outer membrane PlpE protein, while not required for virulence, was surface exposed and could elicit a protective immune response when delivered to chickens in denatured form. Accordingly, it was assumed that linear epitopes are sufficient to induce immunity (Hatfaludi et al., 2012).

In this report, we identified the polytope region of the PlpE protein that is common to all serotypes, thereby offering the possibility of designing a new vaccine candidate to induce protection against all P. multocida serotypes.

Materials and Methods

In silico studies

The known amino acid sequences of PlpE proteins were obtained from the National Center for Biotechnology Information (NCBI) database. Alignment was performed using Multiple Alignment of Vector NTI Advance (TM) 11.0 (December 15, 2008) Invitrogen corporation. Sequences of different serotypes of P. multocida PlpE proteins were used to identify probable epitopes, using Bepipred Linear Epitope Prediction of Immune Epitope Database and Analysis Resource. Potentially antigenic regions were identified by the Kolaskar and Tongaonkar method (1990) using the antigenic peptide prediction tool of immunomedicine group (http://imed.med.ucm.es/Tools/antigenic.html). Potential hydrophilicity was assessed by the Kyte and Doolittle method using the ProtScale tool in the ExPASy database. The half-life was predicted using the ProtParam tool within SIB ExPASy. Linear epitopes of PlpE protein reported to be potent enough to raise immune response against P. multocida infection. The solubility of recombinant proteins was predicted by using the biotech.ou.edu website tool.

Construction of recombinant plasmids and protein purification

The Pasteurella multocida serotype A1, X73, was obtained from the Commercially available Vaccine and Serum Research Institute of Iran (Karaj, Iran). Genomic DNA was extracted by the High Pure PCR Template Preparation Kit (Roche, Germany) and stored at −20°C, then amplified by the PCR method (Singh et al., 2010). The amplified plpE gene was then sequenced by Bioneer (Daejeon, Korea). Three different segments were amplified separately. Fragments 1 and 2 overlapped by SOEing PCR to construct a fusion form (1 + 2) without linker due to separated epitopes. Then, the latter DNA fragment was mixed with fragment 3 and fused using the SOEing PCR program. The final product of overlap PCR contained Segments 1 + 2 + 3 as a fused form called PlpE1 + 2 + 3. PCR products of PlpE-Total, PlpE1 + 2 + 3, and pBAD/gIII A vectors were digested using NcoI and XhoI restriction enzymes for ligation of digested PCR fragments and vector. Competent Top 10 (Novagen, Germany) Escherichia coli cells were prepared, and transformed cells plated on selective medium containing appropriate antibiotics (100 μg/mL ampicillin or 30 μg/mL kanamycin). Recombinant E. coli Top 10 was grown in Luria Broth containing 0.002% L-arabinose (Merck, Germany) for the induction of gene expression. Induced cultures were suspended in binding buffer containing 8 M urea and freeze thawed. Ni-NTA (Qiagen) columns were used for the purification of fusion proteins, then monitored by glycine sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and confirmed by western blotting.

Animal studies

Female, 8-week-old BALB/c mice were used for animal experiments in seven groups. One hundred micrograms of recombinant PlpE, plpE1 + 2 + 3 proteins with or without alum adjuvant (1 mg/mL), and 100 μL commercially available inactivated fowl cholera vaccine were inoculated subcutaneously into the mice at days 0 and 14. Mice in the two negative control groups received either phosphate-buffered saline (PBS) or alum adjuvant only. Tail vein sampling was performed an hour before both booster vaccination and challenge (days 0, 14, and 21, respectively). The sera were maintained at −20°C until further use. Animals were challenged intraperitoneally (IP) with 10 LD50 of Pasteurella multocida A:1, X-73 strain (50 CFU) in 100 μL of saline solution. Survivors were recorded daily for 2 weeks and severely ill mice euthanized. Animal experiments were performed under the approval of the Ethics Committee on Animal Experimentation, Pasteur Institute of Iran, Iran.

Determination of antibody response to vaccine formulations

Specific total immunoglobulin G (IgG) antibody isotype titers were measured using purified recombinant PlpE, PlpE1 + 2 + 3 proteins, and commercially available inactivated fowl cholera vaccine. For determination of antibody levels, these proteins were used as coating antigens at 1 μg/well for PlpE and PlpE1 + 2 + 3 and 4–5 × 10⁹ bacteria for commercially available inactivated fowl cholera vaccine.

Murine sera (1:100 dilution) were used in duplicates as the primary antibody, and the rabbit anti-mouse IgG conjugated to horse radish peroxidase (Sigma, USA) was used as the secondary antibody (1:30,000 dilution). 3,3′,5,5′-tetramethylbenzidine (TMB) substrate [TMB (Pasteur Institute of Iran, Iran)] was used as a colorimetric reagent. Optical density was measured at 405 nm.

Results

In silico studies

The 11 serotypes of P. multocida, which had complete PlpE sequences, were obtained from the NCBI database. Conserved and divergent regions of 11 different PlpEs were evaluated and conserved and similar regions were selected for further evaluation. All possible epitopes of the selected PlpEs of P. multocida were investigated and linear epitopes predicted and selected. Predicted antigenicity of the selected PlpEs of P. multocida was evaluated and all antigenic regions identified. Hydrophilic and hydrophobic regions of the 11 selected PlpEs of P. multocida were identified. Based on these analyses, three regions were of interest as immunodominant regions and were designated region 1 (aa 23–104), region 2 (aa 135–202), and region 3 (aa 262–301) as shown in Fig. 1.

FIG. 1.

Three selected regions within Pasteurella multocida strain ATCC 12948 lipoprotein E (PlpE) gene.

The three selected fragments were assembled in different configurations to make six different polytopes. Due to the accessibility of regions, we decided not to put a linker between them. The predicted solubility of different P. multocida PlpEs and various polytopes was evaluated; all PlpEs and polytopes were insoluble, except for region 3, which was predicted to be 100% soluble. The predicted half-life of different P. multocida PlpEs and fusion proteins was evaluated. All of the PlpEs were predicted to be stable, while the most stable fusion proteins were region 1 plus region 2 and plus region 3 sequentially. The main epitope within region 3 was very unstable in terms of predicted half-life but still 100% soluble.

Construction of recombinant plasmids and protein purification

Overlap PCR using different primers for 5′ and 3′ end of each segment produced fusion segments 1 + 2 and (1 + 2) +3 fragments that were used for cloning. pBAD/gIII A vector, including the gene of interest, was transformed into Top 10 competent E. coli. Colony PCR and digestion using NcoI and XhoI were used to confirm the five best single colonies containing the PlpE-Total and PlpE1 + 2 + 3. Selected colonies were sequenced for confirmation of genes of interest. To produce His-tagged fusion proteins, recombinant Top 10 E. coli were grown in Luria-Bertani containing 0.002 mM L-arabinose, which was assessed as the optimal concentration for expression of both PlpE-Total and PlpE1 + 2 + 3. The presence and purity of the recombinant proteins were visualized on SDS–polyacrylamide gel by Coomassie Blue staining and confirmed by western blot analysis. Western blot analysis using antibodies against His-tag showed that these antibodies reacted with purified recombinant proteins.

Immune responses against PlpE and PlpE1 + 2 + 3 proteins and their protective efficacies

As shown in Fig. 2, serum anti-PlpE/anti-PlpE1 + 2 + 3 IgG level in mice vaccinated with PlpE and PlpE1 + 2 + 3 significantly (p < 0.05) increased after first and second immunizations, as compared with the control mice that received PBS or alum alone. On the other hand, the increase in IgG level following formulation with alum adjuvant in contrast to formulations without alum adjuvant was also significant (p < 0.05).

FIG. 2.

Measurement of IgG and IgG isotypes in the vaccinated mice. Sera were collected at day 0 (before first injection), 14 (2 weeks after first injection), and 28 (2 weeks after second injection) from immunized mice with either PlpE1 + 2 + 3 or PlpE protein with or without alum as well as from control mice (PBS and Alum). ELISA was used for quantitation of the total IgG titers and its isotypes (n = 10–13 for each group). Serum IgG isotype titer was assayed only after second injection. The error bars indicate the interquartile range of each data set. Significant differences between the data sets are marked (p < 0.05; one way ANOVA, Student's t-test). ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; PBS, phosphate-buffered saline.

The protective efficacy of PlpE by the IP challenge of the immunized mice with 10-LD50 of live Pasteurella multocida A:1, X-73 strain is shown in Table 1. Vaccine formulation composed of PlpE admixed with alum adjuvant conferred 69% protection (p = 0.0016) as compared with 31% protection for mice vaccinated with PlpE alone (p = 0.104). Furthermore, protection rate of PlpE1 + 2 + 3 fusion protein formulated with and without alum adjuvant was 69% (p = 0.0016) and 38% (p = 0.046), respectively, after a 2-week daily basis evaluation.

Table 1.

Protection Rate of PlpE1 + 2 + 3 and PlpE Proteins of Pasteurella multocida

Groups	Description	Death within 1 week	Protection rate %
PBS	Control	10/10	0
Alum	Control	10/10	0
(PlpE1 + 2 + 3) + Alum	Alum adjuvanted polytope fusion protein	4/13	69 (p = 0.0016)
PlpE1 + 2 + 3	polytope fusion protein	8/13	38 (p = 0.046)
PlpE + Alum	Alum adjuvanted PlpE protein	4/13	69 (p = 0.0016)
PlpE	PlpE protein	9/13	31 (p = 0.104)
Killed vaccine	Control	0/10	100

PBS, phosphate-buffered saline.

Discussion

Based on our in silico findings, the fusion polytope PlpE1 + 2 + 3 protein showed promise as a potential vaccine candidate. Findings of this study based on challenge results support this notion.

Pasteurellosis is one of the major infections in farm and wild animals, affecting various animals, such as swine, cattle, rabbits, and poultry (Du et al., 2017). P. multocida is the opportunistic pathogen responsible for this contagious disease. While about 5% of world human population is involved in the meat industry, almost 100% of world's population is in contact with animals or their products. The potential for animal to human transmission is thus ever present, especially in the case of poor control of zoonotic disease causative agents.

Currently, the first- and second-generation pasteurellosis vaccines are present in the market. The bacterins of P. multocida provide poor protection against heterologous serotypes, whereas the attenuated vaccines may revert to virulence and cause outbreaks (Okerman and Devriese, 1987, Dowling et al., 2004). It is, therefore, urgent to develop novel vaccine formulations, which are effective, safe, have low production costs, and stimulate cross-protection against heterologous serotypes (Du et al., 2017).

Generally, bacterial outer membrane proteins (OMPs) are highly immunogenic and contain surface-exposed epitopes, which are important for conferring protective immunity in a range of infection models. Previous studies suggested that identifying OMPs may be important in the development of novel vaccine candidates and diagnostic antigens (Dennehy and McClean, 2013). Subunit vaccines made of OMPs can be based on recombinant proteins that have been shown to contain protective epitopes. B cell epitopes have an important role in vaccine development (Hansson et al., 2000) against extracellular bacteria, such as P. multocida. Previous studies determined that the avian P. multocida PlpE protein was a cross-protective antigen, which is located in the P. multocida outer membrane (Hatfaludi et al., 2012). Epitope identification provides valuable information that is essential for understanding antigen components (Al-Hasani et al., 2007). Fusion proteins containing conserved, surface-exposed epitopes thus make attractive vaccine candidates because they may stimulate improved protection against different P. multocida strains.

Similar to other studies (Wilson and Ho, 2013), PlpE1 + 2 + 3 fusion protein with or without alum adjuvant, stimulated specific IgG responses in mice, compared with the commercially available inactivated fowl cholera vaccine and control groups vaccinated by PBS or alum only.

Protection rates of PlpE1 + 2 + 3 with or without alum adjuvant were 38% (p = 0.046) and 69% (p = 0.0016), respectively, against 10-LD50+ challenge with A:1 serotype, X-73 strain. Previous studies showed protection rate for PlpE protein from 70% to 100% (Hatfaludi et al., 2012, Wu et al., 2007), whereas it was 31% (p = 0.104) and 69% (p = 0.0016) in the present study after vaccination with or without alum adjuvant, respectively. Different protection results may be due to differences in strain virulence, mouse strain, protein preparation, or other unknown factors.

The protection results suggest that other antigens are involved in eliciting protection or possibly that cell-mediated immunity may also play a role. The biological function of PlpE remains elusive and it seems the protein can stimulate protection against experimental challenge with P. multocida in both soluble and denatured urea-solubilized forms. Furthermore, this antigen can stimulate up to 100% protection against the homologous strain and significant 50% protection against a second highly virulent strain (Hatfaludi et al., 2012). While the main goal we are looking for is to prove the potential of designed polytope on raise of the immune response of different hosts against pasteurellosis, this rate of protection is acceptable. These results also confirm that IgG level is not a reliable indicator of protection. This fact is studied by Li et al. (2000) and concluded that it is more related to the chronic form of the disease.

In this report, we showed that fusion polytope made of PlpE is a potential candidate in case of protection, but additional studies are needed to verify the protective immunity of PlpE1 + 2 + 3 fusion protein in different animals and to evaluate its potential for cross-protective immunity.

This study is designed in three different parts. The first phase was to find the best candidate part of PlpE protein, construct the gene cassette, produce the protein, analyze it by in silico tools, and prove that it has acceptable protection, which has been done. So, the future direction of this study would be serotype independence study of a candidate protein consisting of evaluation of protection potential of candidate protein on different hosts by different serotypes, evaluation of the potential of candidate protein to raise the cell mediated immunity arm of immunity, and cytokine assays of both Th1- and Th2-mediated immune response. These studies will improve the results, and if successful, which we strongly believe it will be, make the protein a valuable vaccine candidate that can be used to vaccinate as a single universal vaccine that protects the different hosts against different serotypes, which would be a great achievement.

Footnotes

Author Disclosure Statement

I declare that none of the authors listed in the article is employed by a government agency that has a primary function other than research and/or education.

Funding Information

This project honorably sponsored financially by the Pasteur Institute of Iran as a Ph.D. dissertation project.

References

Ahmad

, Rammah

, Sheweita

, Haroun

, et al. Development of immunization trials against Pasteurella multocida . Vaccine, 2014; 32:909–917.

Al-Hasani

, Boyce

, McCarl

, Bottomley

, et al. Identification of novel immunogens in Pasteurella multocida . Microb Cell Fact, 2007; 6:1–5.

Dennehy

, McClean

. Immunoproteomics: The key to discovery of new vaccine antigens against bacterial respiratory infections. Curr Protein Pept Sci, 2013; 13:807–815.

Dowling

, Hodgson

, Dagleish

, Eckersall

, et al. Pathophysiological and immune cell responses in calves prior to and following lung challenge with formalin-killed Pasteurella multocida biotype A: 3 and protection studies involving subsequent homologous live challenge. Vet Immunol Immunopathol, 2004; 100:197–207.

, Wu

, Li

, Fang

, et al. Two novel cross-protective antigens for bovine Pasteurella multocida . Mol Med Rep, 2017; 16:4627–4633.

Farahani

, Esmaelizad

, Jabbari

. Investigation of iron uptake and virulence gene factors (fur, tonB, exbD, exbB, hgbA, hgbB1, hgbB2 and tbpA) among isolates of Pasteurella multocida from Iran. Iran J Microbiol, 2019; 11:191–197.

Hansson

, Nygren

P-Å

, Ståhl

. Design and production of recombinant subunit vaccines. Biotechnol Appl Biochem, 2000; 32:95.

Hatfaludi

, Al-Hasani

, Gong

, Boyce

, et al. Screening of 71 P. multocida proteins for protective efficacy in a fowl cholera infection model and characterization of the protective antigen PlpE. PLoS One, 2012; 7:e39973.

Hofacre

, Glisson

. A serotypic survey of Pasteurella multocida isolated from poultry. Avian Dis, 1986; 30:632.

10.

Hopkins

, Huang

BCTHM

, Olsonc

. Differentiating Turkey Postvaccination Isolants of Pasteurella multocida using arbitrarily primed polymerase chain reaction. Avian Dis, 2016; 42:265–274.

11.

Kolaskar Prasad

, Tongaonkar

. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett, 1990; 276:172–174.

12.

, Nestor

, Saif

, Anderson

, et al. Serum immunoglobulin G and M concentrations did not appear to be associated with resistance to Pasteurella multocida in a large-bodied turkey line and a randombred control population. Poult Sci, 2000; 79:163–166.

13.

Luo

, Vermeij

, Arnoldus

, Jacobs

. (2005). Pasteurella multocida vaccine. Available at https://patents.google.com/patent/WO2006122586A1/en

14.

Mariana

, Hirst

. The immunogenicity and pathogenicity of Pasteurella multocida isolated from poultry in Indonesia. Vet Microbiol, 2000; 72:27–36.

15.

Okerman

, Devriese

. Failure of oil adjuvants to enhance immunity induced in mice by an inactivated rabbit Pasteurella multocida vaccine. Vaccine, 1987; 5:315–318.

16.

Peng

, Wang

, Zhou

, Chen

, et al. Pasteurella multocida: Genotypes and genomics. Microbiol Mol Biol Rev, 2019; 83:e00014-19.

17.

Roier

, Fenninger

, Leitner

, Rechberger

, et al. Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles. Int J Med Microbiol, 2013; 303:247–256.

18.

Singh

, Singh

, Ranjan

, Gupta

, et al. Molecular heterogeneity of plpE gene in Indian isolates of Pasteurella multocida and expression of recombinant PlpE in vaccine strain of P. multocida serotype B: 2. J Vet Sci, 2010; 11:227–233.

19.

Ujvári

, Weiczner

, Deim

, Terhes

, et al. Comparative immunology, microbiology and infectious diseases characterization of Pasteurella multocida strains isolated from human infections. Comp Immunol Microbiol Infect Dis, 2019; 63:37–43.

20.

Wilson

, Ho

. Pasteurella multocida: From Zoonosis to cellular microbiology. Clin Microbiol Rev, 2013; 26:631–655.

21.

, Shien

, Shieh

, Chen

, et al. Protective immunity conferred by recombinant Pasteurella multocida lipoprotein E (PlpE). Vaccine,, 2007; 25:4140–4148.