Development of a novel recombinant influenza vaccine in insect cells

Influenza is a highly contagious viral respiratory illness which is best prevented through vaccination. Currently, all US licensed influenza vaccines are produced in embryonated chicken eggs. The Baculovirus Expression Vector System (BEVS) technology offers several advantages over existing technology, including an exact match between the circulating virus and the antigen in the vaccine, speed, safety, versatility, and reliable scale-up. The expresSF+ insect cells are grown in the absence of serum and have been extensively qualified for safety according to ICH and US FDA guidance and for suitability for the production of recombinant proteins using BEVS. FluBlok^®, a recombinant hemagglutinin influenza vaccine, is composed of purified hemagglutinin protein produced using the BEVS technology. FluBlok has been shown to be safe, effective, and efficacious in human clinical studies.     

Induction of unnatural immunity: prospects for a broadly protective universal influenza vaccine

The immune system normally responds to influenza virus by making neutralizing antibodies to regions of the viral spike, the hemagglutinin, that vary year to year. This natural response protects against circulating subtypes but necessitates production of new vaccines annually. Newer vaccine approaches have succeeded in eliciting broadly neutralizing antibodies to highly conserved yet vulnerable regions of the hemagglutinin and suggest potential pathways for the development of universal influenza vaccines.     

CAP, a new human suspension cell line for influenza virus production

Forced by major drawbacks of egg-based influenza virus production, several studies focused on the establishment and optimization of cell-based production systems. Among numerous possible host cell lines from duck, monkey, canine, chicken, mouse, and human origin, only a few will meet regulatory requirements, accomplish industrial standards, and result in high virus titers. From primary virus isolation up to large-scale manufacturing of human vaccines, however, the most logical choice seems to be the use of human cell lines. For this reason, we evaluated the recently established CAP cell line derived from human amniocytes for its potential in influenza virus production in suspension culture in small scale shaker flask and stirred tank bioreactor experiments. Different human and animal influenza viruses could be adapted to produce hemagglutination (HA) titers of at least 2.0 log₁₀ HA units/100 μL without further process optimization. Adjusting trypsin activity as well as infection conditions (multiplicity of infection, infection medium) resulted in HA titers of up to 3.2 log₁₀ HA units/100 μL and maximum cell-specific virus productivities of 6,400 virions/cell (for human influenza A/PR/8/34 as a reference). Surface membrane expression of sialyloligosaccharides as well as HA N-glycosylation patterns were characterized. Overall, experimental results clearly demonstrate the potential of CAP cells for achieving high virus yields for different influenza strains and the option to introduce a highly attractive fully characterized human cell line compliant with regulatory and industrial requirements as an alternative for influenza virus vaccine production.     

FluBlok,a next generation influenza vaccine manufactured in insect cells

FluBlok, a recombinant trivalent hemagglutinin (rHA) vaccine produced in insect cell culture using the baculovirus expression system, provides an attractive alternative to the current egg-based trivalent inactivated influenza vaccine (TIV). Its manufacturing process presents the possibility for safe and expeditious vaccine production. FluBlok contains three times more HA than TIV and does not contain egg-protein or preservatives. The high purity of the antigen enables administration at higher doses without a significant increase in side-effects in human subjects.The insect cell–baculovirus production technology is particularly suitable for influenza where annual adjustment of the vaccine is required. The baculovirus–insect expression system is generally considered a safe production system, with limited growth potential for adventitious agents. Still regulators question and challenge the safety of this novel cell substrate as FluBlok continues to advance toward product approval. This review provides an overview of cell substrate characterization for expresSF cell line used for the manufacturing of FluBlok.In addition, this review includes an update on the clinical development of FluBlok. The highly purified protein vaccine, administered at three times higher antigen content than TIV, is well tolerated and results in stronger immunogenicity, a long lasting immune response and provides cross-protection against drift influenza viruses.     

Low Seroprevalent Species D Adenovirus Vectors as Influenza Vaccines

Seasonal and pandemic influenza remains a constant threat. While standard influenza vaccines have great utility, the need for improved vaccine technologies have been brought to light by the 2009 swine flu pandemic, highly pathogenic avian influenza infections, and the most recent early and widespread influenza activity. Species C adenoviruses based on serotype 5 (AD5) are potent vehicles for gene-based vaccination. While potent, most humans are already immune to this virus. In this study, low seroprevalent species D adenoviruses Ad26, 28, and 48 were cloned and modified to express the influenza virus A/PR/8/34 hemagglutinin gene for vaccine studies. When studied in vivo, these species D Ad vectors performed quite differently as compared to species C Ad vectors depending on the route of immunization. By intramuscular injection, species D vaccines were markedly weaker than species C vaccines. In contrast, the species D vaccines were equally efficient as species C when delivered mucosally by the intranasal route. Intranasal adenovirus vaccine doses as low as 10⁸ virus particles per mouse induced complete protection against a stringent lethal challenge dose of influenza. These data support translation of species D adenoviruses as mucosal vaccines and highlight the fundamental effects of differences in virus tropism on vaccine applications.     

Oral Delivery of a Novel Attenuated Salmonella Vaccine Expressing Influenza A Virus Proteins Protects Mice against H5N1 and H1N1 Viral Infection

Attenuated strains of invasive enteric bacteria, such as Salmonella, represent promising gene delivery agents for nucleic acid-based vaccines as they can be administrated orally. In this study, we constructed a novel attenuated strain of Salmonella for the delivery and expression of the hemagglutinin (HA) and neuraminidase (NA) of a highly pathogenic H5N1 influenza virus. We showed that the constructed Salmonella strain exhibited efficient gene transfer activity for HA and NA expression and little cytotoxicity and pathogenicity in mice. Using BALB/c mice as the model, we evaluated the immune responses and protection induced by the constructed Salmonella-based vaccine. Our study showed that the Salmonella-based vaccine induced significant production of anti-HA serum IgG and mucosal IgA, and of anti-HA interferon-γ producing T cells in orally vaccinated mice. Furthermore, mice orally vaccinated with the Salmonella vaccine expressing viral HA and NA proteins were completely protected from lethal challenge of highly pathogenic H5N1 as well as H1N1 influenza viruses while none of the animals treated with the Salmonella vaccine carrying the empty expression vector with no viral antigen expression was protected. These results suggest that the Salmonella-based vaccine elicits strong antigen-specific humoral and cellular immune responses and provides effective immune protection against multiple strains of influenza viruses. Furthermore, our study demonstrates the feasibility of developing novel attenuated Salmonella strains as new oral vaccine vectors against influenza viruses.     

Immunogenicity and safety of H1N1 influenza hemagglutinin protein expressed in a baculovirus system

Although most influenza vaccines are produced in eggs, new types of vaccines must be developed. In this study, the immunogenicity and safety of a baculovirus‐expressed hemagglutinin (HA) of H1N1 influenza virus (Korea/01/2009; designated “HA‐Bac‐K”) was compared with those of a commercially available baculovirus‐expressed HA (designated “HA‐Bac‐C”) and an Escherichia coli‐expressed HA (designated “HA‐E. Coli‐K”). HA‐Bac‐K succeeded in inducing hemagglutination inhibition and neutralization antibodies in mouse and ferret models. The different immunogenicities observed may be attributable to the different expression systems and purification protocols used. Our work suggests that HA expressed in a baculovirus system is an effective and safe candidate influenza vaccine.     

Highly conserved M2e and hemagglutinin epitope-based recombinant proteins induce protection against influenza virus infection

Highly pathogenic influenza viruses continue to cause serious threat to public health due to their pandemic potential, calling for an urgent need to develop effective, safe, convenient, and universal vaccines against influenza virus infection. In this study, we constructed two recombinant protein vaccines, 2H5M2e-2H7M2e-H5FP-H7FP (hereinafter M2e-FP-1) and 2H5M2e-H5FP-2H7M2e-H7FP (hereinafter M2e-FP-2), by respectively linking highly conserved sequences of two molecules of ectodomain of M2 (M2e) and one molecule of fusion peptide (FP) epitope of hemagglutinin (HA) of H5N1 and H7N9 influenza viruses in different orders. The Escherichia coli-expressed M2e-FP-1 and M2e-FP-2 proteins induced similarly high-titer M2e-FP-specific antibodies in the immunized mice. Importantly, both proteins were able to prevent lethal challenge of heterologous H1N1 influenza virus, with significantly reduced viral titers and alleviated pathological changes in the lungs, as well as increased body weight and complete survivals, in the challenge mice. Taken together, our study demonstrates that highly conserved M2e and FP epitope of HA of H5N1 and H7N9 influenza viruses can be used as important targets for development of safe and economical universal influenza vaccines, and that the position of H7N9 M2e and H5N1 HA epitope sequences in the vaccine components has no significant effects on the immunogenicity and efficacy of M2e-FP-based subunit vaccines.     

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

Background

The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.

Methodology/Principal Findings

We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.

Conclusion/Significance

This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.     

High accumulation in tobacco seeds of hemagglutinin antigen from avian (H5N1) influenza

Tobacco seeds can be used as a cost effective system for production of recombinant vaccines. Avian influenza is an important respiratory pathogen that causes a high degree of mortality and becomes a serious threat for the poultry industry. A safe vaccine against avian flu produced at low cost could help to prevent future outbreaks. We have genetically engineered tobacco plants to express extracellular domain of hemagglutinin protein from H5N1 avian influenza virus as an inexpensive alternative for production purposes. Two regulatory sequences of seed storage protein genes from Phaseolus vulgaris L. were used to direct the expression, yielding 3.0 mg of the viral antigen per g of seeds. The production and stability of seed-produced recombinant HA protein was characterized by different molecular techniques. The aqueous extract of tobacco seed proteins was used for subcutaneous immunization of chickens, which developed antibodies that inhibited the agglutination of erythrocytes after the second application of the antigen. The feasibility of using tobacco seeds as a vaccine carrier is discussed.     

Plant-produced recombinant influenza vaccine based on virus-like HBc particles carrying an extracellular domain of M2 protein

Conventional influenza vaccines are based on a virus obtained in chicken embryos or its components. The high variability of the surface proteins of influenza virus, hemagglutinin and neuraminidase, requires strain-specific vaccines matching the antigenic specificity of newly emerging virus strains to be developed. A recombinant vaccine based on a highly conservative influenza virus protein M2 fused to a nanosized carrier particle can be an attractive alternative to traditional vaccines. We have constructed a recombinant viral vector based on potato X virus that provides for expression in the Nicotiana benthamiana plants of a hybrid protein M2eHBc consisting of an extracellular domain of influenza virus M2 protein (M2e) fused to hepatitis B core antigen (HBc). This vector was introduced into plant cells by infiltrating leaves with agrobacteria carrying the viral vector. The hybrid protein M2eHBc was synthesized in the infected N. benthamiana plants in an amount reaching 1–2% of the total soluble protein and formed virus-like particles with the M2e peptide presented on the surface. Methods of isolation and purification of M2eHBc particles from plant producers were elaborated. Experiments on mice have shown a high immunogenicity of the plant-produced M2eHBc particles and their protective effect against lethal influenza challenge. The developed transient expression system can be used for production of M2e-based candidate influenza vaccine in plants.     

Single-dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent H5N1, H3N2 and H1N1 viruses

Background

The sudden emergence of novel influenza viruses is a global public health concern. Conventional influenza vaccines targeting the highly variable surface glycoproteins hemagglutinin and neuraminidase must antigenically match the emerging strain to be effective. In contrast, “universal” vaccines targeting conserved viral components could be used regardless of viral strain or subtype. Previous approaches to universal vaccination have required protracted multi-dose immunizations. Here we evaluate a single dose universal vaccine strategy using recombinant adenoviruses (rAd) expressing the conserved influenza virus antigens matrix 2 and nucleoprotein.

Methodology/Principal Findings

In BALB/c mice, administration of rAd via the intranasal route was superior to intramuscular immunization for induction of mucosal responses and for protection against highly virulent H1N1, H3N2, or H5N1 influenza virus challenge. Mucosally vaccinated mice not only survived, but had little morbidity and reduced lung virus titers. Protection was observed as early as 2 weeks post-immunization, and lasted at least 10 months, as did antibodies and lung T cells with activated phenotypes. Virus-specific IgA correlated with but was not essential for protection, as demonstrated in studies with IgA-deficient animals.

Conclusion/Significance

Mucosal administration of NP and M2-expressing rAd vectors provided rapid and lasting protection from influenza viruses in a subtype-independent manner. Such vaccines could be used in the interval between emergence of a new virus strain and availability of strain-matched vaccines against it. This strikingly effective single-dose vaccination thus represents a candidate off-the-shelf vaccine for emergency use during an influenza pandemic.     

Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli

Background

The 23-amino acid extracellular domain of matrix 2 protein (M2e) and the internal nucleoprotein (NP) of influenza are highly conserved among viruses and thus are promising candidate antigens for the development of a universal influenza vaccine. Various M2e- or NP-based DNA or viral vector vaccines have been shown to have high immunogenicity; however, high cost, complicated immunization procedures, and vector-specific antibody responses have restricted their applications. Immunization with an NP–M2e fusion protein expressed in Escherichia coli may represent an alternative strategy for the development of a universal influenza vaccine.

Methodology/Principal Findings

cDNA encoding M2e was fused to the 3′ end of NP cDNA from influenza virus A/Beijing/30/95 (H3N2). The fusion protein (NM2e) was expressed in E. coli and isolated with 90% purity. Mice were immunized with recombinant NM2e protein along with aluminum hydroxide gel and/or CpG as adjuvant. NM2e plus aluminum hydroxide gel almost completely protected the mice against a lethal (20 LD₅₀) challenge of heterologous influenza virus A/PR/8/34.

Conclusions/Significance

The NM2e fusion protein expressed in E. coli was highly immunogenic in mice. Immunization with NM2e formulated with aluminum hydroxide gel protected mice against a lethal dose of a heterologous influenza virus. Vaccination with recombinant NM2e fusion protein is a promising strategy for the development of a universal influenza vaccine.     

Immunosilencing a Highly Immunogenic Protein Trimerization Domain

Many therapeutic proteins and protein subunit vaccines contain heterologous trimerization domains, such as the widely used GCN4-based isoleucine zipper (IZ) and the T4 bacteriophage fibritin foldon (Fd) trimerization domains. We found that these domains induced potent anti-IZ or anti-Fd antibody responses in animals when fused to an HIV-1 envelope glycoprotein (Env) immunogen. To dampen IZ-induced responses, we constructed an IZ domain containing four N-linked glycans (IZN4) to shield the underlying protein surface. When fused to two different vaccine antigens, HIV-1 Env and influenza hemagglutinin (HA), IZN4 strongly reduced the antibody responses against the IZ, but did not affect the antibody titers against Env or HA. Silencing of immunogenic multimerization domains with glycans might be relevant for therapeutic proteins and protein vaccines.     

Live bivalent vaccine for parainfluenza and influenza virus infections

Influenza and human parainfluenza virus infections are of both medical and economical importance. Currently, inactivated vaccines provide suboptimal protection against influenza, and vaccines for human parainfluenza virus infection are not available, underscoring the need for new vaccines against these respiratory diseases. Furthermore, to reduce the burden of vaccination, the development of multivalent vaccines is highly desirable. Thus, to devise a single vaccine that would elicit immune responses against both influenza and parainfluenza viruses, we used reverse genetics to generate an influenza A virus that possesses the coding region for the hemagglutinin/neuraminidase ectodomain of parainfluenza virus instead of the influenza virus neuraminidase. The recombinant virus grew efficiently in eggs but was attenuated in mice. When intranasally immunized with the recombinant vaccine, all mice developed antibodies against both influenza and parainfluenza viruses and survived an otherwise lethal challenge with either of these viruses. This live bivalent vaccine has obvious advantages over combination vaccines, and its method of generation could, in principle, be applied in the development of a "cocktail" vaccine with efficacy against several different infectious diseases.     

Protection of mice against lethal infection with highly pathogenic H7N7 influenza A virus by using a recombinant low-pathogenicity vaccine strain

In 2003, an outbreak of highly pathogenic avian influenza occurred in The Netherlands. The avian H7N7 virus causing the outbreak was also detected in 88 humans suffering from conjunctivitis or mild respiratory symptoms and one person who died of pneumonia and acute respiratory distress syndrome. Here we describe a mouse model for lethal infection with A/Netherlands/219/03 isolated from the fatal case. Because of the zoonotic and pathogenic potential of the H7N7 virus, a candidate vaccine carrying the avian hemagglutinin and neuraminidase proteins produced in the context of the high-throughput vaccine strain A/PR/8/34 was generated by reverse genetics and tested in the mouse model. The hemagglutinin gene of the recombinant vaccine strain was derived from a low-pathogenicity virus obtained prior to the outbreak from a wild mallard. The efficacy of a classical nonadjuvanted subunit vaccine and an immune stimulatory complex-adjuvanted vaccine was compared. Mice receiving the nonadjuvanted vaccine revealed low antibody titers, lack of clinical protection, high virus titers in the lungs, and presence of virus in the spleen, liver, kidneys, and brain. In contrast, mice receiving two doses of the immune stimulatory complex-adjuvanted vaccine revealed high antibody titers, clinical protection, approximately 1,000-fold reduction of virus titers in the lungs, and rare detection of the virus in other organs. This is the first report of an H7 vaccine candidate tested in a mammalian model. The data presented suggest that vaccine candidates based on low-pathogenicity avian influenza A viruses, which can be prepared ahead of pandemic threats, can be efficacious if an effective adjuvant is used.     

Enhanced growth of influenza vaccine seed viruses in vero cells mediated by broadening the optimal pH range for virus membrane fusion

Vaccination is one of the most effective preventive measures to combat influenza. Prospectively, cell culture-based influenza vaccines play an important role for robust vaccine production in both normal settings and urgent situations, such as during the 2009 pandemic. African green monkey Vero cells are recommended by the World Health Organization as a safe substrate for influenza vaccine production for human use. However, the growth of influenza vaccine seed viruses is occasionally suboptimal in Vero cells, which places limitations on their usefulness for enhanced vaccine production. Here, we present a strategy for the development of vaccine seed viruses with enhanced growth in Vero cells by changing an amino acid residue in the stem region of the HA2 subunit of the hemagglutinin (HA) molecule. This mutation optimized the pH for HA-mediated membrane fusion in Vero cells and enhanced virus growth 100 to 1,000 times in the cell line, providing a promising strategy for cell culture-based influenza vaccines.     

Expression in Plants of a Recombinant Protein Based on Flagellin Linked to Conservative Fragments of M2 Protein and Hemagglutintin of Influenza Virus

The composition of traditional influenza vaccines require nearly annual updates due to the high variability of the influenza virus. The use of conservative viral antigens, the extracellular domain of the transmembrane protein M2, and fragments of the second subunit of hemagglutinin provides the opportunity to create recombinant broad-spectrum vaccines. Bacterial flagellin was used as a mucosal adjuvant to increase the immunogenicity of these conservative antigens. Recombinant proteins based on flagellin simultaneously containing M2e and a fragment of the hemagglutinin stem region were expressed in Nicotiana benthamiana plants using a self-replicating vector based on the potato virus X genome. Methods for their isolation from plants and purification have been developed. The developed expression system can be used to produce a new candidate influenza vaccine in plants.