<i>In Silico</i>Identification of Highly Conserved Epitopes of Influenza A H1N1, H2N2, H3N2, and H5N1 with Diagnostic and Vaccination Potential

Muñoz-Medina, José Esteban; Sánchez-Vallejo, Carlos Javier; Méndez‐Tenorio, Alfonso; Monroy-Muñoz, Irma Eloísa; Angeles-Martínez, Javier; Coy-Arechavaleta, Andrea Santos; Santacruz-Tinoco, Clara Esperanza; González-Ibarra, Joaquín; Anguiano-Hernández, Yu-Mei; González-Bonilla, César; Ramón-Gallegos, Eva; Díaz-Quiñonez, José Alberto

doi:10.1155/2015/813047

Cited by 20 publications

(20 citation statements)

References 61 publications

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“…In silico studies of fusion proteins based on FimH from UPEC and MrpH from Proteus mirabilis have revealed that the sequence order is critical for stability and folding (Habibi et al, 2015a). Bioinformatics tools were recently used to propose potential vaccine designs for Helicobacter pylori , enterotoxigenic E. coli, Brucella , influenza viruses, and UPEC (Nazarian et al, 2012; Savar et al, 2014; Golshani et al, 2015; Muñoz-Medina et al, 2015; Habibi et al, 2015b; Mohammad et al, 2016). Additionally, vaccines have been designed to contain T-cell and B-cell immune epitopes, and a class II MHC peptide was also identified using bioinformatics tools (De Groot et al, 2002; De Groot and Moise, 2007).…”

Section: Discussionmentioning

confidence: 99%

Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli

Reyes‐Grajeda

Cruz‐Córdova

Saldaña-Ahuactzi

et al. 2016

Front. Cell. Infect. Microbiol.

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Urinary tract infections (UTIs) are associated with high rates of morbidity and mortality worldwide, and uropathogenic Escherichia coli (UPEC) is the main etiologic agent. Fimbriae assembled on the bacterial surface are essential for adhesion to the urinary tract epithelium. In this study, the FimH, CsgA, and PapG adhesins were fused to generate biomolecules for use as potential target vaccines against UTIs. The fusion protein design was generated using bioinformatics tools, and template fusion gene sequences were synthesized by GenScript in the following order fimH-csgA-papG-fimH-csgA (fcpfc) linked to the nucleotide sequence encoding the [EAAAK]5 peptide. Monomeric (fimH, csgA, and papG), dimeric (fimH-csgA), and trimeric (fimH-csgA-papG) genes were cloned into the pLATE31 expression vector and generated products of 1040, 539, 1139, 1442, and 2444 bp, respectively. Fusion protein expression in BL21 E. coli was induced with 1 mM IPTG, and His-tagged proteins were purified under denaturing conditions and refolded by dialysis using C-buffer. Coomassie blue-stained SDS-PAGE gels and Western blot analysis revealed bands of 29.5, 11.9, 33.9, 44.9, and 82.1 kDa, corresponding to FimH, CsgA, PapG, FC, and FCP proteins, respectively. Mass spectrometry analysis by MALDI-TOF/TOF revealed specific peptides that confirmed the fusion protein structures. Dynamic light scattering analysis revealed the polydispersed state of the fusion proteins. FimH, CsgA, and PapG stimulated the release of 372–398 pg/mL IL-6; interestingly, FC and FCP stimulated the release of 464.79 pg/mL (p ≤ 0.018) and 521.24 pg/mL (p ≤ 0.002) IL-6, respectively. In addition, FC and FCP stimulated the release of 398.52 pg/mL (p ≤ 0.001) and 450.40 pg/mL (p ≤ 0.002) IL-8, respectively. High levels of IgA and IgG antibodies in human sera reacted against the fusion proteins, and under identical conditions, low levels of IgA and IgG antibodies were detected in human urine. Rabbit polyclonal antibodies generated against FimH, CsgA, PapG, FC, and FCP blocked the adhesion of E. coli strain CFT073 to HTB5 bladder cells. In conclusion, the FC and FCP proteins were highly stable, demonstrated antigenic properties, and induced cytokine release (IL-6 and IL-8); furthermore, antibodies generated against these proteins showed protection against bacterial adhesion.

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Section: Discussionmentioning

confidence: 99%

Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli

Reyes‐Grajeda

Cruz‐Córdova

Saldaña-Ahuactzi

et al. 2016

Front. Cell. Infect. Microbiol.

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“…The obtained sequence for NP antigen contained 3 fully homologous conserved epitopes that had been found by Muñoz-Medina J.E. et al [ 20 ]. The NP sequence also contained a few additional highly conserved epitopes: specific for humans, conserved among influenza viruses H1N1, H3N2, H5N1, was epitope ILRGSVAHK; conserved among influenza viruses H1N1, H3N2, H5N1, specific for birds, was epitope KTGGPIYRR, as well as additional less-conserved epitopes.…”

Section: Resultsmentioning

confidence: 99%

Vaccination potential of B and T epitope-enriched NP and M2 against Influenza A viruses from different clades and hosts

et al. 2018

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To avoid outbreaks of influenza virus epidemics and pandemics among human populations, modern medicine requires the development of new universal vaccines that are able to provide protection from a wide range of influenza A virus strains. In the course of development of a universal vaccine, it is necessary to consider that immunity must be generated even against viruses from different hosts because new human epidemic virus strains have their origins in viruses of birds and other animals. We have enriched conserved viral proteins–nucleoprotein (NP) and matrix protein 2 (M2)—by B and T-cell epitopes not only human origin but also swine and avian origin. For this purpose, we analyzed M2 and NP sequences with respect to changes in the sequences of known T and B-cell epitopes and chose conserved and evolutionarily significant epitopes. Eventually, we found consensus sequences of M2 and NP that have the maximum quantity of epitopes that are 100% coincident with them. Consensus epitope-enriched amino acid sequences of M2 and NP proteins were included in a recombinant adenoviral vector. Immunization with Ad5-tet-M2NP induced strong CD8 and CD4 T cells responses, specific to each of the encoded antigens, i.e. M2 and NP. Eight months after immunization with Ad5-tet-M2NP, high numbers of M2- and NP-responding “effector memory” CD44posCD62neg T cells were found in the mouse spleens, which revealed a long-term T cell immune memory conferred by the immunization. In all, the challenge experiments showed an extraordinarily wide-ranging efficacy of protection by the Ad5-tet-M2NP vaccine, covering 5 different heterosubtypes of influenza A virus (2 human, 2 avian and 1 swine).

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“…The extracellular domain of M2e is highly conserved among influenza A virus subtypes (Muñoz-Medina et al 2015;Zebedee and Lamb 1988); thus, it was adopted as a target antigen in the development of a universal influenza vaccine. In the present study, a single M2e induced a poor M2e-specific humoral response (Fig.…”

Section: Discussionmentioning

confidence: 99%

Improving Cross-Protection against Influenza Virus Using Recombinant Vaccinia Vaccine Expressing NP and M2 Ectodomain Tandem Repeats

et al. 2019

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Conventional influenza vaccines need to be designed and manufactured yearly. However, they occasionally provide poor protection owing to antigenic mismatch. Hence, there is an urgent need to develop universal vaccines against influenza virus. Using nucleoprotein (NP) and extracellular domain of matrix protein 2 (M2e) genes from the influenza A virus A/Beijing/30/95 (H3N2), we constructed four recombinant vaccinia virus-based influenza vaccines carrying NP fused with one or four copies of M2e genes in different orders. The recombinant vaccinia viruses were used to immunize BALB/C mice. Humoral and cellular responses were measured, and then the immunized mice were challenged with the influenza A virus A/Puerto Rico/8/34 (PR8). NP-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length NP, while robust M2e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of M2e. All recombinant viruses elicited NP-and M2e-specific cellular immune responses in mice. Only immunization with RVJ-4M2eNP induced remarkably higher levels of IL-2 and IL-10 cytokines specific to M2e. Furthermore, RVJ-4M2eNP immunization provided the highest cross-protection in mice challenged with 20 MLD 50 of PR8. Therefore, the cross-protection potentially correlates with both NP and M2e-specific humoral and cellular immune responses induced by RVJ-4M2eNP, which expresses a fusion antigen of full-length NP preceded by four M2e repeats. These results suggest that the rational fusion of NP and multiple M2e antigens is critical toward inducing protective immune responses, and the 4M2eNP fusion antigen may be employed to develop a universal influenza vaccine.

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In SilicoIdentification of Highly Conserved Epitopes of Influenza A H1N1, H2N2, H3N2, and H5N1 with Diagnostic and Vaccination Potential

Cited by 20 publications

References 61 publications

Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli

Dimeric and Trimeric Fusion Proteins Generated with Fimbrial Adhesins of Uropathogenic Escherichia coli

Vaccination potential of B and T epitope-enriched NP and M2 against Influenza A viruses from different clades and hosts

Improving Cross-Protection against Influenza Virus Using Recombinant Vaccinia Vaccine Expressing NP and M2 Ectodomain Tandem Repeats

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