Edited by: Arun Kumar, GlaxoSmithKline (GSK) Vaccines, Italy
Reviewed by: Wouter Koudstaal, Janssen Prevention Center, Netherlands; Kaissar Tabynov, Research Institute for Biological Safety Problems, Kazakhstan
This article was submitted to Immunotherapies and Vaccines, a section of the journal Frontiers in Immunology.
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Influenza virus is a negative segmented RNA virus without DNA intermediate. This makes it safer as a vaccine delivery vector than most DNA viruses that have potential to integrate their genetic elements into host genomes. In this study, we developed a universal influenza viral vector, expressing the receptor binding subdomain of botulinum neurotoxin A (BoNT/A). We tested the growth characters of the engineered influenza virus in chicken eggs and Madin–Darby canine kidney epithelial cells (MDCK), and showed that it can be produced to a titer of 5 × 106 plaque forming unites/ml in chicken eggs and MDCK cells. Subsequently, mice intranasally vaccinated with the engineered influenza virus conferred protection against challenge with lethal doses of active BoNT/A toxin and influenza virus. Our results demonstrated the feasibility to develop a dual purpose nasal vaccine against both botulism and influenza.
Influenza A virus is a member of family orthomyxoviridae, which contains a segmented RNA genome. With the elucidation of replication mechanism of influenza virus, generation of influenza virus from DNA became feasible and extended the research of influenza dramatically. The most important application of this system is creation of live-attenuated vaccine or the generation of influenza vector to express foreign antigens (
Botulinum neurotoxins (BoNTs, A–H) are the most poisonous substances known in nature. They may be used as bioterrorism agents or in biological warfare. Currently, there is no FDA licensed botulism vaccine available for public use. Natural BoNT is divided into a light chain (L) and a heavy chain (H) (
Madin–Darby canine kidney epithelial cells (MDCK) were cultured in modified Eagle’s medium with 10% fetal calf serum. 293T cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal calf serum. BALB/c mice were purchased from the Jackson Laboratories (Bar Harbor, ME, USA). Animal experimental protocol (protocol number is 10020) was approved by the Institutional Animal Care and Use Committee at Texas Tech University Health Sciences Center, and carried out in accordance with the US Public Health Service Guide for the Care and Use of Laboratory Animals (NRC Publication, 8th ed.) and other related federal statutes and regulations of the Animal Welfare Act. MLD50 were determined by Reed–Muench method (
First, we synthesized a gene segment coding 2A peptide followed by a multiple cloning site (mcs) (
About 100 pfu viruses were inoculated into allantoic cavity of a 9-day-old specific pathogen free (SPF) embryonated eggs (Charles River, CT, USA). At 72 h post-inoculation, viruses were harvested and titrated on monolayer MDCK cells by using the standard plaque assay method. To measure virus growth curve, monolayer MDCK cells were initially infected with virus at multiplicity of infection (MOI) of 0.001. After 1 h incubation, virus was removed and MDCK cells were washed with PBS supplemented with Ca++ and Mg++, then medium were replaced with fresh medium supplemented with 0.2% BSA and 1 μg/ml Tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin. At 24, 36, 48, 60, and 72 h post-infection, virus samples were collected, respectively, and titrated on monolayer MDCK cells in triplicate.
Viral RNA was extracted from virus samples using RNeasy kit (Qiagen, CA, USA), and subjected to the RT-PCR amplification by using SuperScript III Reverse Transcriptase kit (Invitrogen, CA, USA). RT-PCR products were analyzed by electrophoresis in 0.8% agarose gel. The images were taken by VersaDoc imager (BioRad, CA, USA).
Madin–Darby canine kidney epithelial cells were infected with influenza virus at MOI of 1. At 8 h post-infection, cells were harvested and lysed. Proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane. Then, membrane was incubated with anti-BoNT/A-HC50 serum (prepared in our lab), rabbit anti-NP polyclonal antibody (Immune-tech), and β-actin antibody, respectively. After being washed, membrane was incubated with goat anti-rabbit or -mice IgG-HRP for 1 h. Finally, membrane was developed with chemiluminescence HRP substrate (Takara Bio Inc.), and images were taken by using ImageQuant Las4000 (GE Health, PA, USA).
Forty-eight (48) 6-week female BALB/c mice were divided to 8 groups. Subsequently, the mice were intranasally vaccinated with 5 pfu influenza virus. At 4 weeks post-infection, mice were boosted with the same dose. Blood samples were collected on the days of immunization and challenge. On day 42, mice were challenged with 10× MLD50 BoNT/A toxin (BEI Resources, cat# NR-4529, NIAID, NIH) or 100× MLD50 PR8 influenza virus. After being challenged, mice were observed every day. In the BoNT/A toxin challenged groups, data of survived mice and dead mice were calculated. In the PR8 challenged groups, mouse weights were recorded every day. When the mouse weight loss was >25%, the mouse was euthanized and sacrificed according the guide of the Institutional Animal Care and Use Committee at Texas Tech University Health Sciences Center.
Briefly, microplates were coated with recombinant BoNT/A receptor binding subdomain overnight. After antigen was removed and plates were blocked, sera were diluted and added into each wells, and incubated at 4oC overnight. One hundred microliters of diluted goat anti-mouse IgG-Fc antibody conjugated to alkaline-phosphtase were added into each well and incubated for 1 h. Subsequently, plates were washed and pNPP substrate was added and incubated for 20 min. After sufficient color development, stop solution was added into each well. Finally, the plates were read in the Gene5 microplate reader (BioTek).
Comparison between virus titers was performed by using
At first, NA gene segment was engineered by inserting a multi-cloning site with a 2A cleavage site. Then a universal replication-competent influenza viral vector was rescued. To demonstrate the use of this universal influenza viral vector, a 618 bp gene segment of BoNT/A HC50-rbsd was inserted into engineered NA gene segment. An engineered influenza virus carrying target gene segment was rescued, purified, and propagated in embryonated chicken eggs. The rescued viruses were named PR8-wt, PR8-mcs, PR8-B/A-rbsd, respectively. Then, viruses propagated in embryonated chicken eggs were harvested, aliquoted, and stored at −80°C for subsequent use.
To confirm rescued influenza viruses carrying anticipated NA gene segment, viral RNA was extracted, and NA gene segments were amplified. Results in Figure
Plaque assay on MDCK cells was performed to analyze the growth characteristics of engineered influenza viruses. In Figure
In Figure
Influenza virus has plenty of advantages that make it worthy of consideration for use as a viral vector for pathogens that are problematic to vaccine development. There are well-established protocols for large-scale production of both live and inactivated influenza viruses, and live influenza vaccines have shown to elicit strong T cell immune response to stimulate mucosal and systemic responses (
Botulinum neurotoxins are the most poisonous substances in the nature and potential bioterrorism agents. With the advantage of molecular technology, identification of non-toxic domains of BoNT toxin provides a useful method to develop a promising vaccine for botulism. In this research, our results showed that the smaller receptor binding subdomain of BoNT/A toxin is an effective antigen to stimulate humoral immune response. Additional research in this direction may lead to a multivalent vaccine against all types of BoNTs using the smaller antigen (HC50-rbsd) instead of HC50 (
JL and MZ designed the research, analyzed the data, and wrote the manuscript. JL, DD-A, and YC performed experiments.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was partially supported by the US Public Service research grant AI072139 and AI055946 (MZ) from the National Institute of Allergy and Infectious Diseases, and by an internal fund from the Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center at El Paso.