Design of a New Vaccine Prototype against Porcine Circovirus Type 2 (PCV2), M. hyopneumoniae and M. hyorhinis Based on Multiple Antigens Microencapsulation with Sulfated Chitosan
Abstract
:1. Introduction
2. Materials and Methods
2.1. Vaccine Microparticles
2.2. Morfoestructural by Scanning Electron Microscopy (SEM) and Elemental Analysis by Energy Dispersive Spectroscopy (EDS) of Vaccine Prototypes
2.3. Analysis of Surface Charge (Zeta Potential) and Polydispersity Index (PdI) of Microparticles of Vaccine Prototypes
2.4. Experimental Design
2.5. Controlled Conditions for Animals
2.6. Experimental Challenge
2.7. Intratracheal Inoculation
2.8. Obtaining and Processing of Samples
2.9. Detection of the Presence of PCV2, M. hyopneumoniae and M. hyorhinis by PCR (Real Time)
2.10. Evaluation of the Induction of Antibodies for PCV2, M. hyopneumoniae and M. hyorhinis through ELISA Test
2.11. Statistical Analysis
3. Results
3.1. Morfoestructural by Scanning Electron Microscopy (SEM) and Elemental Analysis by Energy Dispersive Spectroscopy (EDS) of Vaccine Prototypes
3.1.1. Elemental Analysis by Energy Dispersive Spectroscopy (EDS)
3.1.2. Analysis of Surface Charge and Polydispersity of Microparticles of Vaccine Prototypes
3.2. Experimental Challenge of Animals under Controlled Conditions
Evaluation of Induction of Specific Antibodies against PCV2 and M. hyopneumoniae and M. hyorhinis by ELISA Test
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Group | Number of Animals | Experimental Treatment |
---|---|---|
1 | 5 | Experimental formula 1 mL (1 mg/mL of MPs)–IM |
2 | 5 | Experimental formula 3 mL (1 mg/mL of MPs)–O/N |
3 | 5 | Placebo–3 mL (1 mg/mL of MPs)–O/N |
4 | 5 | Commercial vaccine 1 mL (1 dose)–IM |
Event and/or Experimental Procedure | Day of the Event in Experimental Period | Duration of the Period (Days) | Number of Week | Sample |
---|---|---|---|---|
Time 0 (Acclimatization) | 1 | 10 | 1,2 | nasal swab/blood |
Time 1/first immunization and start of the experiment (administration of experimental formulations, placebo and commercial vaccine for each treatment) | 11 | 14 | 2,3 | nasal swab/blood |
Time 2 | 25 | 7 | 4 | nasal swab/blood |
Vaccine boosters (21 days after the 1st immunization) | 31 | 7 | 5 | - |
Time 3 | 38 | 7 | 6 | nasal swab/blood |
Experimental challenge | 45 | 7 | 7 | - |
Time 4/Vaccine boosters (42 days after the 1st immunization) | 52 | 7 | 8 | nasal swab/blood |
Time 5 | 68 | 16 | 9,10,11 | nasal swab/blood |
Time 6 (necropsy) | 80 | 12 | 11,12 | Bronchial swab/inguinal lymph node/blood |
Primers | Sequence |
---|---|
Fw PCV2 | 5′-GGGATGATCTACTGAGACTGTGTGA-3′ |
Rv PCV2 | 5′-GGGGAAAGGGTGACGAACT-3′ |
Probe PCV2 | 5′-/56-FAM/AATGGTACT/ZEN/CCTCAACTGCTGTCCCAGC/3IABkFQ/-3′ |
Fw M. hyop | 5′-GTCAAAGTCAAAGTCAGCAAAC-3′ |
Rv M. hyop | 5′-AGCTGTTCAAATGCTTGTCC-3′ |
Probe M. hyop | 5′-/5Cy5/ACCAGTTTC/TAO/CACTTCATCGCCTCA/3IAbRQSp/-3′ |
Fw M. hyor | 5′-CGGGATGTAGCAATACATTCAG-3′ |
Rv M. hyor | 5′-AGAGGCATGATGATTTGACGTC-3′ |
PCV2/M. hyopneumoniae | M. hyorhinis | |
---|---|---|
Method | Real Time qPCR Multiplex | Real Time PCR (Sybr Green) |
Temperature | 95 °C | 95 °C |
Time | 3 min | 3 min |
Cycles | 40 (95 °C for 15 s and 60 °C per 1 min) | 40 (95 °C for 15 s and 60 °C for 35 s) |
Element | Concentration (%) | |
---|---|---|
ChS + PRC-Antigens | Chc + PRC-Antigens | |
C | 41.35 | 68.01 |
N | 9.68 | 6.81 |
O | 44.4 | 25.17 |
S | 4.56 | 0.02 |
Total | 100 | 100 |
MPs + Antigen | Zeta Potential (mV) | Polydispersity (PdI) |
---|---|---|
Chc + M. hyorhinis | 48.2 | 0.595 |
ChS + M. hyorhinis | −21.7 | 0.314 |
Chc + M. hyopneumoniae | 44.7 | 0.712 |
ChS + M. hyopneumoniae | −38.8 | 0.210 |
Chc + PCV2 | 58.2 | 0.680 |
ChS + PCV2 | −39.1 | 0.293 |
Samples Obtained in the Post-Mortem Experimental Period (T6) during the Necropsy | ||||||
---|---|---|---|---|---|---|
Bronchial Swab | Inguinal Lymph Node | Sera | ||||
Pathogen | PCV2 | M. hyop. | M. hyor. | PCV2 | PCV2 | |
Animal (ID) | ||||||
1 | − | − | − | + | − | |
2 | − | + | + | + | − | |
3 | + | − | − | + | − | |
4 | + | − | + | + | − | |
5 | − | − | − | − | − | |
6 | + | − | + | − | − | |
7 | − | − | + | + | − | |
8 | − | − | − | − | − | |
9 | + | + | + | + | − | |
10 | − | + | + | + | − | |
11 | − | − | − | − | − | |
12 | + | − | − | + | − | |
13 | − | − | − | + | − | |
14 | + | − | + | − | − | |
15 | − | − | + | − | − | |
16 | − | − | + | + | − | |
17 | + | + | − | + | − | |
18 | − | − | − | + | − | |
19 | + | − | + | − | − | |
20 | − | − | − | − | − |
Pathogen | PCV2 | M. hyopneumoniae | M. hyorhinis | |
---|---|---|---|---|
Group | ||||
(1) Experimental formula (IM) | 1/5 | 1/5 | 0/5 | |
(2) Experimental formula (O/N) | 5/5 | 0/5 | 3/5 | |
(3) Placebo control (O/N) | 3/5 | 3/5 | 3/5 | |
(4) Commercial vaccine control (IM) | 3/5 | 0/5 | 3/5 |
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© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Arrieta-Mendoza, D.; Garces, B.; Hidalgo, A.A.; Neira, V.; Ramirez, G.; Neira-Carrillo, A.; Bucarey, S.A. Design of a New Vaccine Prototype against Porcine Circovirus Type 2 (PCV2), M. hyopneumoniae and M. hyorhinis Based on Multiple Antigens Microencapsulation with Sulfated Chitosan. Vaccines 2024, 12, 550. https://doi.org/10.3390/vaccines12050550
Arrieta-Mendoza D, Garces B, Hidalgo AA, Neira V, Ramirez G, Neira-Carrillo A, Bucarey SA. Design of a New Vaccine Prototype against Porcine Circovirus Type 2 (PCV2), M. hyopneumoniae and M. hyorhinis Based on Multiple Antigens Microencapsulation with Sulfated Chitosan. Vaccines. 2024; 12(5):550. https://doi.org/10.3390/vaccines12050550
Chicago/Turabian StyleArrieta-Mendoza, Darwuin, Bruno Garces, Alejandro A. Hidalgo, Victor Neira, Galia Ramirez, Andrónico Neira-Carrillo, and Sergio A. Bucarey. 2024. "Design of a New Vaccine Prototype against Porcine Circovirus Type 2 (PCV2), M. hyopneumoniae and M. hyorhinis Based on Multiple Antigens Microencapsulation with Sulfated Chitosan" Vaccines 12, no. 5: 550. https://doi.org/10.3390/vaccines12050550