A prime/boost vaccination with HA DNA and Pichia-produced HA protein elicits a strong humoral response in chickens against H5N1
Introduction
Vaccination is the major tool for the prevention and control of influenza. Due to the low efficacy of current seasonal influenza vaccines, and by a continuous threat of new pandemic strain outbreaks, there is a pressing need for new generation influenza vaccines. To overcome these difficulties, novel vaccine strategies have been developed (Spackman and Swayne, 2013). Since antibodies that neutralize the influenza virus are mainly directed against the hemagglutinin (HA) protein, much research has focused on this antigen, and a dynamic development of subunit vaccines based on HA currently occurs. Indeed, influenza vaccine candidates utilizing recombinant technology and various expression systems (e.g. bacteria, baculovirus, plant or mammalian cells) are under development or in clinical trials (Buckland et al., 2014, Le Mauff et al., 2015, Moresco et al., 2010, Verma et al., 2012). An additional benefit of using subunit vaccines is their appliance for the vaccination in the buffer zone during H5N1 outbreaks, since they enable the differentiation of infected individuals from those who have been vaccinated. DNA vaccination, which is a variant of the subunit vaccine approach, is a promising new strategy that offers several advantages. The plasmids used for DNA vaccination are usually propagated in E. coli and can be easily purified in large scale by ion exchange chromatography, which makes this approach economically attractive. Moreover, it has been suggested that priming with an influenza DNA vaccine received long before pandemic attack could present significant benefits, including reducing the amount of target vaccine needed and conferring some initial immunity level in human populations (Lu, 2011). Numerous researchers have demonstrated that H5N1 DNA vaccines have a protective effect in model animals (Liu, 2011, Meunier et al., 2016, Stachyra et al., 2014a). However, despite the great potential of this type of vaccine, some reports have concluded that DNA plasmids used alone fail to induce significant protection against pathogens in large animals and humans, even with the use of various molecular adjuvants (Lu, 2009).
The efficacy of DNA vaccines can be augmented by boosting the vaccinated individuals with either protein or viral vector vaccines. Such combined prime/boost immunizations have been successfully exploited to improve the breadth of the cellular and humoral immune response elicited by various vaccines against different viral pathogens in animal studies, including HIV (Pal et al., 2006), HCV (Li et al., 2006), HPV (Radaelli et al., 2012) and bacterial (Shkreta et al., 2004) or parasitic diseases (Mazumder et al., 2011). Very good effects have been described for DNA vaccines used in combined prime/boost immunization strategies against influenza using inactivated virus (Wang et al., 2008), live-attenuated influenza viruses (Suguitan et al., 2011), virus like particles (Ding et al., 2011, Lin et al., 2012) and recombinant proteins (Luo et al., 2012). Most studies using recombinant antigens were performed in mice, which are a relevant and widespread animal model, but are not a natural host for avian influenza viruses. Studies with natural host species, such as chickens, are needed to evaluate these novel experimental vaccine formulations. Additionally, recombinant proteins produced in human, insect or bacterial cells are usually used for the combined DNA-protein immunizations. Recently, several phase I clinical studies have reported surprisingly good results of approaches with HA-DNA vaccines used as primers and inactivated corresponding viruses as boosters (Crank et al., 2015, Ledgerwood et al., 2015a, Ledgerwood et al., 2015b, Ledgerwood et al., 2013). The results of these experiments confirm the potential of the combined DNA/protein immunization approach and emphasize the need to develop novel components for such an approach. However, the production methods mentioned above possess some limitations, e.g. vaccine antigen produced on cell lines have to be thoroughly screened for viruses and/or potential cancerogenic agents and/or protein contamination, which might cause adverse effects. One of the systems used for recombinant influenza vaccine manufacturing is baculovirus expression system based on insect cells. However, this solution involves high manufacturing costs connected with the demand for high qualified staff and expensive reagents. Therefore, new, inexpensive and safe methods of producing vaccine antigens are still being explored. Despite the existing solutions that have emerged so far, there is a constant need to develop a new method of vaccine antigen and new antigen production.
The Pichia pastoris system has been extensively utilized as an industrial platform to produce various biopharmaceuticals, including vaccines (Shanvac™, Elovac™, Gavac™), however we are not aware of any reports on using it for the DNA/protein prime/boost strategy. Pichia cells are able to carry out post-translational modifications, offer the possibility of producing even gram amounts of recombinant protein per liter of culture, and in addition in a secretory fashion which greatly facilitates subsequent purification of desired protein.
In this study we evaluated the effects of application of the subunit prime/boost vaccine strategy against HPAI H5N1 in chickens. Although each of the subunit vaccines applied in this work has been tested individually against HPAI H5N1 viruses, the combination of both vaccines in a prime/boost strategy has not been previously reported. Here we primed chickens with a DNA plasmid encoding H5 HA from the HPAI virus (A/swan/Poland/305-135V08/2006 (H5N1)) and boosted once with H5 oligomers from the same strain, produced in P. pastoris. A comparison of the antibody levels and HI titers elicited with DNA/DNA, DNA/protein and protein/protein prime/boost strategies indicated that the level of antibodies capable of hemagglutinin inhibition was significantly higher in DNA/protein than in protein/protein vaccinated animals, while the difference between the DNA/DNA and DNA/protein groups was not significant.
Section snippets
Plasmid used for DNA vaccination
A plasmid containing the cDNA encoding full length (except the 341-RRRKKR-347 residues, a proteolytic cleavage site between HA1 and HA2 subunits) hemagglutinin (HA) from A/swan/Poland/305-135V08/2006 (H5N1); clade 2.2 and optimized to the domestic chicken codon bias was described earlier (Stachyra et al., 2014b, Stachyra et al., 2016)
Recombinant H5 HA protein expression, purification and analysis
The same H5N1 virus strain used as the source of the DNA vaccine was also used as the source of cDNA encoding HA cloned into the P. pastoris expression vector. A
Characterization of recombinant hemagglutinin protein used as a subunit vaccine
Recombinant H5 HA protein was produced in P. pastoris cells as a secreted protein. Such an approach proved to be efficient, convenient and economically justified since a high level of purity was achieved after one-step purification via Ni-based chromatography (Fig. 1A). The apparent size of the protein (about 70 kDa) was consistent with the molecular weight of recombinant glycosylated HA. Purified antigen fractions produced by the immobilized metal affinity chromatography (IMAC) procedure were
Discussion
The traditional influenza vaccine production is a lengthy procedure. It requires the use of eggs and requires the generation of virus reassortants, which can effectively multiply in hen‘s eggs to obtain a sufficient yield of vaccine product. Moreover, the production process depends on a continuous supply of eggs, which has always represented a bottleneck in this traditional approach. The next generation vaccines, such as recombinant HA protein–based or DNA–based subunit vaccines, have become
Authors’ contribution
ASt, AP and AG-S prepared the DNA vaccine and conducted the immunization experiments, MP, AM and EK prepared the recombinant protein, MO, KŚ and ZM designed and performed the HI experiments. All authors participated in study design and data analysis, manuscript and figures preparation, have read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Ethics approval and consent to participate
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The experiments with chickens were approved by the Second Local Ethical Committee for Animal Experiments at the Medical University of Warsaw, Permit Number 17/2009.
Acknowledgments
We dedicate this work to the memory of Professor Włodzimierz Zagórski-Ostoja, who was actively involved in its initial stage. This work was realized in frame of the Polish Vaccine Consortium (PVC) and it was supported by Innovative Economy Program, Grant No. WND-POIG.01.01.02-00-007/08, by Grant No. PBS2/A7/14/2014 from the National Centre for Research and Development and by KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal- Safe Food,” decision of Ministry of
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