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Paragraph: Mucosal Vaccination  with Recombinant Lactobacillus casei-Displayed CTA1-Conjugated Consensus Matrix Protein-2 (sM2) Induces Broad Protection against Divergent Influenza Subtypes in BALB/c Mice

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979752/

SHA: efaa556b484fbcd9cc34832ffac53ef3e834e9c0

Authors: Chowdhury, Mohammed Y. E.; Li, Rui; Kim, Jae-Hoon; Park, Min-Eun; Kim, Tae-Hwan; Pathinayake, Prabuddha; Weeratunga, Prasanna; Song, Man Ki; Son, Hwa-Young; Hong, Seung-Pyo; Sung, Moon-Hee; Lee, Jong-Soo; Kim, Chul-Joong
Date: 2014-04-08
DOI: 10.1371/journal.pone.0094051
License: cc-by

Abstract: To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with (pgsA-CTA1-sM2/L. casei) or without (pgsA-sM2/L. casei) cholera toxin subunit A1 (CTA1) on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD(50) of A/EM/Korea/W149/06(H5N1), A/Puerto Rico/8/34(H1N1), A/Aquatic bird /Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005(H7N3), and A/Chicken/Korea/116/2004(H9N2) viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes. Text:   remains most economical and effective means against respiratory diseases caused by influenza viruses [1] . Based on the circulating viruses in the population, trivalent vaccine strains have been developed and are used for the influenza virus protection [2] . The most acceptable current available strategy is the intramuscular administration of inactivated vaccines produced by egg-based manufacturing systems which while effective, are hampered by limited capacity and flexibility [3] . However, vaccine strains must be frequently adapted to match the circulating viruses throughout the world [4] . In addition, the levels of antibody induced by the inactivated vaccine have been observed to decrease by 75% over an 8-month period [2, 5] . Therefore, alternative strategies for developing broadly cross-protective, safe and effective vaccines against influenza viral infections are of prominent importance. Matrix protein 2 (M2) is highly conserved among influenza A virus strains, indicating that M2 is an attractive target for developing a universal vaccine [6] . In previous studies, various constructs of the M2 vaccine have been developed and tested, including recombinant Escherichia coli (E. coli) expressing M2 fusion protein, adenoviral vectors expressing the M2 protein, plasmid DNA encoding M2 [7] [8] [9] and peptides encoding M2e [11] , each of which was able to elicit protective immune responses in mice. However, the drawback of these M2-based vaccines is their low immunogenicity; additionally, most of them would require intramuscular injections. Therefore, many strategies have been applied focusing on increasing the immunogenicity of M2-based vaccines, for example, fusion of M2 with different carrier molecules like human papilloma virus L protein [12] , keyhole limpet hemocyanin [10] and flagellin [13] . Furthermore, vaccinations with different adjuvants and routes of administration have been applied to evaluate their protection against divergent strains of influenza viruses. Mice immunized mucosally with an M2 or virus like particles (VLPs) adjuvanted with cholera toxin (CT) demonstrated better protection compared to mice subjected to parenteral immunization [14, 15] . However, due to the adverse effects of CT in humans, investigators have attempted to identify nontoxic subunits with adjuvanticity by removing either subunit A or subunit B [16] . E. coli expressing cholera toxin subunit A1 (CTA1) fused with the D-fragment of Staphylococcus aureus showed the adjuvant effects without any reactogenicity of the A1 subunit in the mucosal vaccine [6] . Although, chemical or genetic conjugation of M2 may not present M2 in its native tetrameric form, extracellularly accessible antigens expressed on the surfaces of bacteria are better recognized by the immune system than those that are intracellular [17] . Thus, choice of delivery vehicle is also an important concern for potential mucosal vaccines. Recently, lactic acid bacteria (LAB) presenting influenza virus antigens have been studied [3, 18, 19] . For mucosal immunization, LAB is a more attractive delivery system than other live vaccine vectors, such as Shigella, Salmonella, and Listeria [20, 21] . It is considered safe and exhibits an adjuvant-like effect on mucosal and systemic immunity [18, 22, 23] . Anchoring of the target protein to the cell surfaces of LAB is primarily intended to use in mucosal vaccines. The transmembrane protein pgsA is one of the poly-cglutamate synthetase complexes of Bacillus subtilis [17, 24, 25] , which is a well-studied anchor protein is able to fuse the target protein to its C terminus and stabilize the complex by anchoring it in the cell membrane. Since sM2 is a highly conserved and promising target for a universal vaccine and CTA1 is strong mucosal adjuvant, in this study, we developed constructs using a consensus sM2 gene reconstituted from the analysis of H1N1, H5N1 and H9N2 influenza viruses (no trans-membrane domain) with or without the fusion of CTA1. To achieve this, we used a novel expression vector that can express a pgsA gene product as an anchoring matrix. Our target antigens, sM2 and CTA1, were displayed on the surface of Lactobacillus casei, and the oral or intranasal administration of recombinant L. casei induced systemic and mucosal immune responses that have the potential to protect against the lethal challenges of divergent influenza subtypes. A total of 672 female BALB/c mice (5 weeks old) were purchased from Samtako (Seoul, Korea) and housed in ventilated cages. The mice were managed with pelleted feed and tap water ad libitum, maintained in a specific-pathogen-free environment and all efforts were made to minimize suffering following approval from the Institutional Animal Care and Use Committee of of Bioleaders Corporation, Daejeon, South Korea, protocol number: BSL-ABLS-13-002. Immunizations of animal were conducted in biosafety level (BSL)-2 laboratory facilities. Mice were divided into 6 experimental sets, each consisting of 2 subsets: 1 for oral and 1 for intranasal administration which contained 4 groups each. Out of 6, 4 sets had 14 mice per group. One sets had 17 (3 mice for lung histopathology and immunohistochemistry), and the last contained 11 mice per group (3 mice for CTL response). Concentrations of recombinant L. casei were determined by colony forming units (CFU). In each subset, 2 groups received 10 10 CFU of pgsA-sM2/L. casei or pgsA-CTA1-sM2/L. casei, and the remaining two groups received the same concentration of pKV-pgsA/L. casei or PBS in 100 ml orally via intragastric lavage at days 0 to 3, 7 to 9 and 21 to 23. Similarly, 10 9 CFU of recombinant cells were administered in 20 ml suspensions into the nostrils of lightly anesthetized mice on days 0 to 3, 7 to 9 and 21. Blood samples were collected from the retro-orbital plexus at days 21, 14 and 28; sera were separated by centrifugation for 5 minutes at 12,0006g and stored at 220uC until analysis. At day 28, 3 mice in each group were randomly sacrificed to collect IgA sample from lungs and intestine and stored at 270uC until analysis. Spleens were collected aseptically at day 28 for the analysis of the CTL response randomly from 3 mice of one set. The rest of the mice from the same set were maintained for 6 months from the date of the last boosting to measure the long-lasting immune responses and protection efficacy. The avian influenza viruses A/EM/Korea/W149/06(H5N1), A/Puerto Rico/8/34(H1N1), A/Aquatic bird/Korea/W81/2005 (H5N2), A/Aquatic bird/Korea/W44/2005(H7N3), and A/ Chicken/Korea/116/2004(H9N2) used in this study were kindly provided by Dr. Young-Ki Choi (College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea). All viruses were propagated in the allantoic fluid of 10-day-old chicken embryos, and 50% mouse lethal doses (MLD 50 ) were determined in 8-week-old naive BALB/ c mice. Ether narcosis-anesthetized mice were intranasally infected with 10 times the MLD 50 of challenge viruses in 20 ml of PBS. Six mice in each group were sacrificed on 3 and 5 dpi to check virus titer in lungs and other 5 mice remained in each group have been used for survival. Mice were monitored every alternate day at fixed time point for measuring the weight loss and survival. Mice were euthanized if moribund, i.e. weight loss, ruffled fur, shivering, tachypnea, respiratory distress, hypothermia and poorly responsive to external stimuli, remaining were considered as survival number. After final monitoring, all the survived mice were humanely euthanized using CO 2 inhalation for 5 minutes. At 180 days after the final vaccination, mice from one set were challenged with H5N2 for measuring the long lasting immune responses. All challenge tests were conducted inside an approved BSL-3+ facility under appropriate conditions. Bacterial Strains and Cloning for the Construction of Recombinant Plasmid PgsA-sM2/L. casei and PgsA-CTA1-sM2/L. casei In this study, E. coli JM83 was used for cloning and L. casei L525 was used for surface expression of the target protein. These bacteria were grown in LB and MRS media, respectively. The plasmid pKV-Pald-PgsA, harboring the pgsA genes of Bacillus subtilis, was used to construct the surface display plasmid, which was a kind gift from the Bioleaders Corporation (Daejeon, South Korea). A gene encoding the consensus sequence of M2 spanning the residues of the extracellular and cytoplasmic domains without the transmembrane domain of influenza virus was generated. The consensus sequences were created based on the most common amino acids in each position of the alignment of H1N1, H5N1 and H9N2; then, they were synthesized and used as templates for the construction of the plasmids pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei by cloning, as described previously [26, 27] . The sM2 gene was modified by adding a Kpn I site at the 59 terminal and Sal I at the 39 terminal for cloning. The polymerase chain reaction (PCR) was performed to amplify the gene using the primer pair 59-GGGGTACCTCATTATTAACA-39, and 59-ACGTCGACT-CATTATTCAAGTTCAATAATG AC-39. Similarly, a BamH I site at the 59 terminal and a Kpn I site at the 39 terminal end were added to the CTA1 gene using primers 59-CGGGATCCAAT-GATGATAAGTTATAT-39 and 59-GGGT ACCCGAT-GATCTTGGAGC ATT-39. The modified genes were ligated into the T Easy Vector (Invitrogen, Seoul, Korea). Genes were then digested with Kpn I-Sal I for sM2 and BamH I-Kpn I for CTA1. The digested sM2 was ligated to the plasmid vector pKV-pgsA for the construction of pKV-pgsA-sM2. Similarly, CTA1 was ligated for the construction of pKV-pgsA-CTA1-sM2. The ligated products were transformed into E. coli JM83 competent cells, as previously described, using an electroporation method [17] . The profiles of the recombinant plasmids were confirmed by restriction endonuclease digestion and DNA sequencing (Solgent, Seoul, Korea). After confirmation, the plasmids were transformed into L. casei L525 by electroporation and named pgsA-sM2/L. casei and pgsA-CTA1-sM2/L. casei. The recombinant L. casei containing pgsA, pgsA-sM2 and pgsA-CTA1-sM2 genes were grown at 30uC for 48 hours. Cells were harvested by centrifugation at 6,0006g for 10 minutes at 4uC, followed by washing two times with sterile phosphate-buffered saline (PBS).

Question: What is the most effective treatment against influenza?
Vaccination