Immunogenic Potency of a Chimeric Protein Comprising InvH and IpaD against Salmonella and Shigella spp

Shigella and Salmonella cause serious problems in many subjects, including young children and the elderly, especially in developing countries. Chimeric proteins carrying immunogens increase immune response. In-silico tools are applied to design vaccine candidates. Invasion plasmid antigens D (ipaD) gene is one of the Shigella virulence factors. The N-terminal region of the IpaD plays a significant role in invading the host cell. Invasion protein H (invH) gene plays important role in bacterial adherence and entry into epithelial cells. A recombinant chimeric construct, containing IpaD and InvH was designed and used as a vaccine candidate against Shigella and Salmonella enteritidis. After bioinformatics assessments, the construct was designed, synthesized, and expressed in E.coli. Chimeric protein, IpaD, and InvH were purified with Ni-NTA chromatography. Purified proteins were confirmed with western blotting and then were injected into separate mice groups. The antibody titer was estimated with an enzyme-linked immunosorbent assay (ELISA). Mice were challenged with 10, 100, and 1000 LD50 of Salmonella, and the sereny test was performed for Shigella. The Codon adaptation index of the chimeric gene was increased to 0.84. Validation results showed that 97.9% of residues lie in the favored or additional allowed region of the Ramachandran plot. A significant antibody rise was observed in all test groups. The immunized mice with chimer and InvH could tolerate 100 LD50 of Salmonella. In the sereny test, the application of bacteria treated with immunized mice sera of both antigens showed no infection in Guinea pigs' eyes. The recombinant protein could protect animal models against Salmonella and Shigella and therefore can be considered as a suitable vaccine candidate against these two pathogens.

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Naturally acquired antibody response to a Plasmodium falciparum chimeric vaccine candidate GMZ2.6c and its components (MSP-3, GLURP, and Pfs48/45) in individuals living in Brazilian malaria-endemic areas

Malaria Journal ◽

10.1186/s12936-021-04020-6 ◽

2022 ◽

Vol 21 (1) ◽

Author(s):

Barbara Oliveira Baptista ◽

Ana Beatriz Lopes de Souza ◽

Evelyn Kety Pratt Riccio ◽

Cesare Bianco-Junior ◽

Paulo Renato Rivas Totino ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Antibody Response ◽

Vaccine Candidate ◽

Chimeric Protein ◽

The State ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Multi Stage ◽

Endemic Areas ◽

Different Levels

Abstract Background The GMZ2.6c malaria vaccine candidate is a multi-stage Plasmodium falciparum chimeric protein which contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, a fusion protein of GLURP and MSP-3, that has been shown to be well tolerated, safe and immunogenic in clinical trials performed in a malaria-endemic area of Africa. However, there is no data available on the antigenicity or immunogenicity of GMZ2.6c in humans. Considering that circulating parasites can be genetically distinct in different malaria-endemic areas and that host genetic factors can influence the immune response to vaccine antigens, it is important to verify the antigenicity, immunogenicity and the possibility of associated protection in individuals living in malaria-endemic areas with different epidemiological scenarios. Herein, the profile of antibody response against GMZ2.6c and its components (MSP-3, GLURP and Pfs48/45) in residents of the Brazilian Amazon naturally exposed to malaria, in areas with different levels of transmission, was evaluated. Methods This study was performed using serum samples from 352 individuals from Cruzeiro do Sul and Mâncio Lima, in the state of Acre, and Guajará, in the state of Amazonas. Specific IgG, IgM, IgA and IgE antibodies and IgG subclasses were detected by Enzyme-Linked Immunosorbent Assay. Results The results showed that GMZ2.6c protein was widely recognized by naturally acquired antibodies from individuals of the Brazilian endemic areas with different levels of transmission. The higher prevalence of individuals with antibodies against GMZ2.6c when compared to its individual components may suggest an additive effect of GLURP, MSP-3, and Pfs48/45 when inserted in a same construct. Furthermore, naturally malaria-exposed individuals predominantly had IgG1 and IgG3 cytophilic anti-GMZ2.6c antibodies, an important fact considering that the acquisition of anti-malaria protective immunity results from a delicate balance between cytophilic/non-cytophilic antibodies. Interestingly, anti-GMZ2.6c antibodies seem to increase with exposure to malaria infection and may contribute to parasite immunity. Conclusions The data showed that GMZ2.6c protein is widely recognized by naturally acquired antibodies from individuals living in malaria-endemic areas in Brazil and that these may contribute to parasite immunity. These data highlight the importance of GMZ2.6c as a candidate for an anti-malarial vaccine.

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Murine Immune Responses to Virus-Like Particle-Associated Pre- and Postfusion Forms of the Respiratory Syncytial Virus F Protein

Journal of Virology ◽

10.1128/jvi.00384-15 ◽

2015 ◽

Vol 89 (13) ◽

pp. 6835-6847 ◽

Cited By ~ 28

Author(s):

Lori McGinnes Cullen ◽

Madelyn R. Schmidt ◽

Sarah A. Kenward ◽

Robert T. Woodland ◽

Trudy G. Morrison

Keyword(s):

Respiratory Syncytial Virus ◽

Neutralizing Antibodies ◽

Vaccine Candidate ◽

Neutralizing Antibody ◽

Enzyme Linked Immunosorbent Assay ◽

Chimeric Proteins ◽

Wild Type ◽

F Protein ◽

Core Proteins ◽

Syncytial Virus

ABSTRACTVirus-like particles (VLPs) built on the Newcastle disease virus (NDV) core proteins, NP and M, and containing two chimeric proteins, F/F and H/G, composed of respiratory syncytial virus (RSV) fusion protein (F) and glycoprotein (G) ectodomains fused to the transmembrane and cytoplasmic domains of the NDV F and HN proteins, respectively, stimulate durable, protective RSV neutralizing antibodies in mice. Here, we report the properties of VLPs constructed to contain mutant RSV F protein ectodomains stabilized in prefusion (pre-F/F) or postfusion (post-F/F) configurations. The structures of the chimeric proteins assembled into VLPs were verified immunologically by their reactivities with a conformationally restricted anti-F protein monoclonal antibody. Following immunization of mice, without adjuvant, pre-F/F-containing VLPs induced significantly higher neutralizing antibody titers than the post-F/F-containing VLPs or the wild-type F/F-containing VLPs after a single immunization but not after prime and boost immunization. The specificities of anti-F IgG induced by the two mutant VLPs were assessed by enzyme-linked immunosorbent assay (ELISA) using soluble forms of the prefusion and postfusion forms of the F protein as targets. While both types of VLPs stimulated similar levels of IgG specific for the soluble postfusion F protein, titers of IgG specific for prefusion F induced by the pre-F/F-containing VLPs were higher than those induced by post-F/F-containing VLPs. Thus, VLPs containing a stabilized prefusion form of the RSV F protein represent a promising RSV vaccine candidate.IMPORTANCEThe development of vaccines for respiratory syncytial virus has been hampered by a lack of understanding of the requirements for eliciting high titers of neutralizing antibodies. The results of this study suggest that particle-associated RSV F protein containing mutations that stabilize the structure in a prefusion conformation may stimulate higher titers of protective antibodies than particles containing F protein in a wild-type or postfusion conformation. These findings indicate that the prefusion F protein assembled into VLPs has the potential to produce a successful RSV vaccine candidate.

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Toxoplasma gondii Tetravalent Chimeric Proteins as Novel Antigens for Detection of Specific Immunoglobulin G in Sera of Small Ruminants

Animals ◽

10.3390/ani9121146 ◽

2019 ◽

Vol 9 (12) ◽

pp. 1146 ◽

Cited By ~ 2

Author(s):

Lucyna Holec-Gąsior ◽

Bartłomiej Ferra ◽

Weronika Grąźlewska

Keyword(s):

Toxoplasma Gondii ◽

Small Ruminants ◽

Chimeric Protein ◽

High Specificity ◽

Elisa Test ◽

Dense Granule ◽

Enzyme Linked Immunosorbent Assay ◽

Chimeric Proteins ◽

Apical Membrane Antigen 1 ◽

Toxoplasma Gondii Infection

The detection of Toxoplasma gondii infection in small ruminants has important significance for public health and veterinary medicine. This study, for the first time, describes the reactivity of four tetravalent chimeric proteins (AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, AMA1-SAG2-GRA1-ROP1, and SAG2-GRA1-ROP1-GRA2) containing immunodominant regions from the AMA1 (apical membrane antigen 1), SAG2 (surface antigen 2), GRA1 (dense granule antigen 1), GRA2 (dense granule antigen 2), and ROP1 (rhoptry antigen 1) with specific IgG antibodies from the sera of small ruminants with the use of an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISA based on a Toxoplasma lysate antigen (TLA). All chimeric proteins were characterized by high specificity (between 96.39% to 100%), whereas the sensitivity of the IgG ELISAs was variable (between 78.49% and 96.77%). The highest sensitivity was observed in the IgG ELISA test based on the AMA1-SAG2-GRA1-ROP1. These data demonstrate that this chimeric protein can be a promising serodiagnostic tool for T. gondii infection in small ruminants.

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Mutation E48K in PB1 Polymerase Subunit Improves Stability of a Candidate Live Attenuated Influenza B Virus Vaccine

Vaccines ◽

10.3390/vaccines9070800 ◽

2021 ◽

Vol 9 (7) ◽

pp. 800

Author(s):

Jongsuk Mo ◽

Stivalis Cardenas-Garcia ◽

Jefferson J. S. Santos ◽

Lucas M. Ferreri ◽

C. Joaquín Cáceres ◽

...

Keyword(s):

Vaccine Candidate ◽

The Elderly ◽

Potential Interaction ◽

Respiratory Pathogen ◽

Influenza B Virus ◽

Influenza B ◽

Epitope Tag ◽

Live Attenuated Vaccine ◽

B Virus ◽

Homologous Challenge

Influenza B virus (IBV) is a major respiratory pathogen of humans, particularly in the elderly and children, and vaccines are the most effective way to control it. In previous work, incorporation of two mutations (E580G, S660A) along with the addition of an HA epitope tag in the PB1 segment of B/Brisbane/60/2008 (B/Bris) resulted in an attenuated strain that was safe and effective as a live attenuated vaccine. A third attempted mutation (K391E) in PB1 was not always stable. Interestingly, viruses that maintained the K391E mutation were associated with the mutation E48K. To explore the contribution of the E48K mutation to stability of the K391E mutation, a vaccine candidate was generated by inserting both mutations, along with attenuating mutations E580G and S660A, in PB1 of B/Bris (B/Bris PB1att 4M). Serial passages of the B/Bris PB1att 4M vaccine candidate in eggs and MDCK indicated high stability. In silico structural analysis revealed a potential interaction between amino acids at positions 48 and 391. In mice, B/Bris PB1att 4M was safe and provided complete protection against homologous challenge. These results confirm the compensatory effect of mutation E48K to stabilize the K391E mutation, resulting in a safer, yet still protective, IBV LAIV vaccine.

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Self-assembling thermostable chimeras as new platform for arsenic biosensing

Scientific Reports ◽

10.1038/s41598-021-82648-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rosanna Puopolo ◽

Ilaria Sorrentino ◽

Giovanni Gallo ◽

Alessandra Piscitelli ◽

Paola Giardina ◽

...

Keyword(s):

Inclusion Bodies ◽

Association Constant ◽

Immobilized Enzyme ◽

Thermus Thermophilus ◽

Chimeric Protein ◽

The Self ◽

Chimeric Proteins ◽

Self Assembling ◽

Langmuir Isotherm Model ◽

Activity Loss

AbstractThe correct immobilization and orientation of enzymes on nanosurfaces is a crucial step either for the realization of biosensors, as well as to guarantee the efficacy of the developed biomaterials. In this work we produced two versions of a chimeric protein, namely ArsC-Vmh2 and Vmh2-ArsC, which combined the self-assembling properties of Vmh2, a hydrophobin from Pleurotus ostreatus, with that of TtArsC, a thermophilic arsenate reductase from Thermus thermophilus; both chimeras were heterologously expressed in Escherichia coli and purified from inclusion bodies. They were characterized for their enzymatic capability to reduce As(V) into As(III), as well as for their immobilization properties on polystyrene and gold in comparison to the native TtArsC. The chimeric proteins immobilized on polystyrene can be reused up to three times and stored for 15 days with 50% of activity loss. Immobilization on gold electrodes showed that both chimeras follow a classic Langmuir isotherm model towards As(III) recognition, with an association constant (KAsIII) between As(III) and the immobilized enzyme, equal to 650 (± 100) L mol−1 for ArsC-Vmh2 and to 1200 (± 300) L mol−1 for Vmh2-ArsC. The results demonstrate that gold-immobilized ArsC-Vmh2 and Vmh2-ArsC can be exploited as electrochemical biosensors to detect As(III).

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Safety and immunogenicity of an mRNA-lipid nanoparticle vaccine candidate against SARS-CoV-2

Wiener klinische Wochenschrift ◽

10.1007/s00508-021-01922-y ◽

2021 ◽

Author(s):

Peter G. Kremsner ◽

Philipp Mann ◽

Arne Kroidl ◽

Isabel Leroux-Roels ◽

Christoph Schindler ◽

...

Keyword(s):

Immune Responses ◽

Vaccine Candidate ◽

Phase 1 ◽

Lipid Nanoparticles ◽

Enzyme Linked Immunosorbent Assay ◽

Receptor Binding Domain ◽

Igg Antibodies ◽

Phase 1 Study ◽

S Protein ◽

Dosage Escalation

Summary Background We used the RNActive® technology platform (CureVac N.V., Tübingen, Germany) to prepare CVnCoV, a COVID-19 vaccine containing sequence-optimized mRNA coding for a stabilized form of SARS-CoV‑2 spike (S) protein encapsulated in lipid nanoparticles (LNP). Methods This is an interim analysis of a dosage escalation phase 1 study in healthy 18–60-year-old volunteers in Hannover, Munich and Tübingen, Germany, and Ghent, Belgium. After giving 2 intramuscular doses of CVnCoV or placebo 28 days apart we assessed solicited local and systemic adverse events (AE) for 7 days and unsolicited AEs for 28 days after each vaccination. Immunogenicity was measured as enzyme-linked immunosorbent assay (ELISA) IgG antibodies to SARS-CoV‑2 S‑protein and receptor binding domain (RBD), and SARS-CoV‑2 neutralizing titers (MN50). Results In 245 volunteers who received 2 CVnCoV vaccinations (2 μg, n = 47, 4 μg, n = 48, 6 μg, n = 46, 8 μg, n = 44, 12 μg, n = 28) or placebo (n = 32) there were no vaccine-related serious AEs. Dosage-dependent increases in frequency and severity of solicited systemic AEs, and to a lesser extent local AEs, were mainly mild or moderate and transient in duration. Dosage-dependent increases in IgG antibodies to S‑protein and RBD and MN50 were evident in all groups 2 weeks after the second dose when 100% (23/23) seroconverted to S‑protein or RBD, and 83% (19/23) seroconverted for MN50 in the 12 μg group. Responses to 12 μg were comparable to those observed in convalescent sera from known COVID-19 patients. Conclusion In this study 2 CVnCoV doses were safe, with acceptable reactogenicity and 12 μg dosages elicited levels of immune responses that overlapped those observed in convalescent sera.

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A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a Trivalent Vaccine

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00640-10 ◽

2010 ◽

Vol 54 (11) ◽

pp. 4750-4757 ◽

Cited By ~ 6

Author(s):

Gaobing Wu ◽

Yuzhi Hong ◽

Aizhen Guo ◽

Chunfang Feng ◽

Sha Cao ◽

...

Keyword(s):

Protective Antigen ◽

Vaccine Candidate ◽

Lethal Factor ◽

Lethal Dose ◽

Chimeric Protein ◽

Dominant Negative ◽

Lethal Challenge ◽

Edema Factor ◽

Trivalent Vaccine

ABSTRACT Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF) to PA and the transportation of LF/EF. Therefore, we replaced PA in LFn-PA with a dominant-negative inhibitory PA (DPA), i.e., PAF427D. In in vitro models of anthrax intoxication, the LFn-DPA chimera showed 3-fold and 2-fold higher potencies than DPA in protecting sensitive cells against anthrax lethal toxin (LeTx) and edema toxin (EdTx), respectively. In animal models, LFn-DPA exhibited strong potency in rescuing mice from lethal challenge with LeTx. We also evaluated the immunogenicity and immunoprotective efficacy of LFn-DPA as an anthrax vaccine candidate. In comparison with recombinant PA, LFn-DPA induced significantly higher levels of the anti-PA immune response. Moreover, LFn-DPA elicited an anti-LF antibody response that could cross-react with EF. Mice immunized with LFn-DPA tolerated a LeTx challenge that was 5 times its 50% lethal dose. Thus, LFn-DPA represents a highly effective trivalent vaccine candidate for both preexposure and postexposure vaccination. Overall, we have developed a novel and dually functional reagent for the prophylaxis and treatment of anthrax.

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PREPARATION AND EVALUATION OF CHEMICALLY INACTIVATED SALMONELLA ENTERITIDIS VACCINE IN CHICKENS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2017.v10i11.20144 ◽

2017 ◽

Vol 10 (11) ◽

pp. 341

Author(s):

Mounir M El-safty ◽

Hala Mahmoud ◽

Eman Sa Zaki ◽

Howaida I Abd-alla

Keyword(s):

Immune Responses ◽

Salmonella Enteritidis ◽

Serum Antibody ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Chemically Induced ◽

Before And After ◽

Group A ◽

Group B ◽

Cell Envelopes

Objective: Salmonella enteritidis ghosts (SEGs) is a non-living empty bacterial cell envelopes which were generated using a different concentration of sodium hydroxide (NaOH) 6.4 mg/mL and evaluated as a vaccine candidate in specific pathogen-free (SPF) chicken. SEGs have been produced by chemical-mediated lysis and evaluated the potential efficacy of chemically induced SEG vaccine and its ability to induce protective immune responses against virulent S. enteritidis challenge in SPF chickens.Methods: SPF chickens were divided into three groups: Group A (non-vaccinated control), Group B (vaccinated with prepared vaccine), and Group C (vaccinated with commercial vaccine).Results: Vaccination of SPF chicken with SEGs induced higher immune responses before and after virulent challenge. SPF chicken vaccinated with SEGs showed increasing in serum enzyme-linked immunosorbent assay (ELISA) antibodies. During the vaccination period, Groups B and C showed higher serum antibody titer compared to Group A. The minimal inhibitory concentration (MIC) of NaOH was capable of inducing non-living SEGs, and it has successfully generated non-living SEGs by MIC of NaOH.Conclusion: It is a one-step process which means easy manufacturing and low production cost compared to protein E-mediated lysis method. Chemically induced SEG vaccine is a highly effective method for inducing protective immunity. This study strongly suggests that SEGs will be a permissive vaccine, as the method of inhibition of S. enteritidis was safe and cheaper than other methods, and it gave a good protection.

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