Integrating high cell density cultures with adapted laboratory evolution for improved Gag-HA virus-like particles production in stable insect cell lines.

Stable insect cell lines are emerging as an alternative to the insect cell-baculovirus expression vector system (IC-BEVS) for protein expression, benefiting from being a virus-free, nonlytic system. Still, the titers achieved are considerably lower. In this study, stable insect (Sf-9 and High Five) cells producing Gag virus-like particles (VLPs) were first adapted to grow under hypothermic culture conditions (22°C instead of standard 27°C), and then pseudotyped with a model membrane protein (influenza hemagglutinin [HA]) for expression of Gag-HA VLPs. Adaptation to lower temperature led to an increase in protein titers of up to 12-fold for p24 (as proxy for Gag-VLP) and sixfold for HA, with adapted Sf-9 cells outperforming High Five cells. Resulting Gag-HA VLPs producer Sf-9 cells were cultured to high cell densities, that is, 100 × 106 cell/ml, using perfusion (ATF® 2) in 1 L stirred-tank bioreactors. Specific p24 and HA production rates were similar to those of batch culture, enabling to increase volumetric titers by 7-8-fold without compromising the assembly of Gag-HA VLPs. Importantly, the antigen (HA) quantity in VLPs generated using stable adapted cells in perfusion was ≈5-fold higher than that from IC-BEVS, with the added benefit of being a baculovirus-free system. This study demonstrates the potential of combining stable expression in insect cells adapted to hypothermic culture conditions with perfusion for improving Gag-HA VLPs production.

Drivers of recombinant soluble influenza A virus hemagglutinin and neuraminidase expression in mammalian cells.

Recombinant soluble trimeric influenza A virus hemagglutinins (HA) and tetrameric neuraminidases (NAs) have proven to be excellent tools to decipher biological properties. Receptor binding and sialic acid cleavage by recombinant proteins correlate satisfactorily compared to whole viruses. Expression of HA and NA can be achieved in a plethora of different laboratory hosts. For immunological and receptor interaction studies however, insect and mammalian cell expressed proteins are preferred due to the presence of N-linked glycosylation and disulfide bond formation. Because mammalian-cell expression is widely applied, an increased expression yield is an important goal. Here we report that using codon-optimized genes and sfGFP fusions, the expression yield of HA can be significantly improved. sfGFP also significantly increased expression yields when fused to the N-terminus of NA. In this study, a suite of different hemagglutinin and neuraminidase constructs are described, which can be valuable tools to study a wide array of different HAs, NAs and their mutants.

Recombinant Haemagglutinin Derived From the Ciliated Protozoan Tetrahymena thermophila Is Protective Against Influenza Infection.

Current influenza vaccines manufactured using eggs have considerable limitations, both in terms of scale up production and the potential impact passaging through eggs can have on the antigenicity of the vaccine virus strains. Alternative methods of manufacture are required, particularly in the context of an emerging pandemic strain. Here we explore the production of recombinant influenza haemagglutinin using the ciliated protozoan Tetrahymena thermophila. For the first time we were able to produce haemagglutinin from both seasonal influenza A and B strains. This ciliate derived material was immunogenic, inducing an antibody response in both mice and non-human primates. Mice immunized with ciliate derived haemagglutinin were protected against challenge with homologous influenza A or B viruses. The antigen could also be combined with submicron particles containing a Nod2 ligand, significantly boosting the immune response and reducing the dose of antigen required. Thus, we show that Tetrahymena can be used as a manufacturing platform for viral vaccine antigens.

SPRi-based hemagglutinin quantitative assay for influenza vaccine production monitoring.

Influenza vaccine manufacturers lack tools, whatever the involved production bioprocess (egg or cell-based), to precisely and accurately evaluate vaccine antigen content from samples. Indeed, the gold standard single-radial immunodiffusion (SRID) assay, which remains the only validated assay for the evaluation of influenza vaccine potency, is criticized by the scientific community and regulatory agencies since a decade for its high variability, lack of flexibility and low sensitivity. We hereby report an imaging surface plasmon resonance (SPRi) assay for the quantification of both inactivated vaccine influenza antigens and viral particles derived from egg- and cell-based production samples, respectively. The assay, based on fetuin-hemagglutinin interactions, presents higher reproducibility (<3%) and a greater analytical range (0.03-20 µg/mL) than SRID for bulk monovalent and trivalent vaccine and its limit of detection was evaluated to be 100 times lower than the SRID's one. Finally, viral particles production through cell culture-based bioprocess was also successfully monitored using our SPRi-based assay and a clear correlation was found between the biosensor response and total virus particle content.

Development of a universal influenza vaccine using hemagglutinin stem protein produced from Pichia pastoris.

The development of a universal influenza vaccine has become a major effort to combat the high mutation rate of influenza. To explore the use of the highly conserved stem region of hemagglutinin (HA) as a universal vaccine, we produced HA-stem-based protein using yeast expression systems. The glycosylation effects on the immunogenicity and protection activities were investigated. The yield of the A/Brisbane/59/2007 HA stem produced from Pichia pastoris reached 100 mg/l. The immunogenicity of HA stem proteins in various glycoforms was further investigated and compared. All glycoforms of the HA stem protein can induce cross-reactive antibody responses, antibody-dependent cellular cytotoxicity (ADCC)-mediated protection as well as T-cell responses, with broad protection in mice. The monoglycosylated form of the A/Brisbane/59/2007 HA stem produced in yeast, together with the glycolipid C34 as the adjuvant, can elicit greater ADCC responses, better neutralizing activities against heterologous strains, and broader protection in mice.

A secretary bi-cistronic baculovirus expression system with improved production of the HA1 protein of H6 influenza virus in insect cells and Spodoptera litura larvae.

A bi-cistronic baculovirus expression vector was constructed to facilitate the expression, detection, and isolation of the hemagglutinin (HA) fragment HA1 of H6N1 avian influenza virus (AIV) in an insect and a culture of its cells. In this construct, the GP67sp signal peptide promoted the secretion of the recombinant protein into the culture medium, and improved protein expression and purification. Enhanced green fluorescent protein, co-expressed through an internal ribosome entry site, served as a visible reporter for protein expression detection. The hemolymph of Spodoptera litura larvae infected with the bi-cistronic baculovirus was collected for the purification of the recombinant HA1, which was found to be glycosylated, and monomeric and trimeric forms of the recombinant HA1 were identified. Proteins expressed in both the cell culture and larvae served as effective subunit vaccines for the production of antiserum against HA. The antiserum recognized the H6 subtype of AIV but not the H5 subtype.

Harnessing an RNA-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation.

A novel protein-folding function of RNA has been recognized, which can outperform previously known molecular chaperone proteins. The RNA as a molecular chaperone (chaperna) activity is intrinsic to some ribozymes and is operational during viral infections. Our purpose was to test whether influenza hemagglutinin (HA) can be assembled in a soluble, trimeric, and immunologically activating conformation by means of an RNA molecular chaperone (chaperna) activity. An RNA-interacting domain (RID) from the host being immunized was selected as a docking tag for RNA binding, which served as a transducer for the chaperna function for de novo folding and trimeric assembly of RID-HA1. Mutations that affect tRNA binding greatly increased the soluble aggregation defective in trimer assembly, suggesting that RNA interaction critically controls the kinetic network in the folding/assembly pathway. Immunization of mice resulted in strong hemagglutination inhibition and high titers of a neutralizing antibody, providing sterile protection against a lethal challenge and confirming the immunologically relevant HA conformation. The results may be translated into a rapid response to a new influenza pandemic. The harnessing of the novel chaperna described herein with immunologically tailored antigen-folding functions should serve as a robust prophylactic and diagnostic tool for viral infections.-Yang, S. W., Jang, Y. H., Kwon, S. B., Lee, Y. J., Chae, W., Byun, Y. H., Kim, P., Park, C., Lee, Y. J., Kim, C. K., Kim, Y. S., Choi, S. I., Seong, B. L. Harnessing an RNA-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation.

Biological Evaluation of Uridine Derivatives of 2-Deoxy Sugars as Potential Antiviral Compounds against Influenza A Virus.

Influenza virus infection is a major cause of morbidity and mortality worldwide. Due to the limited ability of currently available treatments, there is an urgent need for new anti-influenza drugs with broad spectrum protection. We have previously shown that two 2-deoxy sugar derivatives of uridine (designated IW3 and IW7) targeting the glycan processing steps during maturation of viral glycoproteins show good anti-influenza virus activity and may be a promising alternative approach for the development of new anti-influenza therapy. In this study, a number of IW3 and IW7 analogues with different structural modifications in 2-deoxy sugar or uridine parts were synthesized and evaluated for their ability to inhibit influenza A virus infection in vitro. Using the cytopathic effect (CPE) inhibition assay and viral plaque reduction assay in vitro, we showed that compounds 2, 3, and 4 exerted the most inhibitory effect on influenza virus A/ostrich/Denmark/725/96 (H5N2) infection in Madin-Darby canine kidney (MDCK) cells, with 50% inhibitory concentrations (IC50) for virus growth ranging from 82 to 100 (µM) without significant toxicity for the cells. The most active compound (2) showed activity of 82 µM with a selectivity index value of 5.27 against type A (H5N2) virus. Additionally, compound 2 reduced the formation of HA glycoprotein in a dose-dependent manner. Moreover, an analysis of physicochemical properties of studied compounds demonstrated a significant linear correlation between lipophilicity and antiviral activity. Therefore, inhibition of influenza A virus infection by conjugates of uridine and 2-deoxy sugars is a new promising approach for the development of new derivatives with anti-influenza activities.

Translational regulation of viral secretory proteins by the 5' coding regions and a viral RNA-binding protein.

A primary function of 5' regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. In this study, we show how the regions coding for the ER-targeting sequences of the influenza glycoproteins NA and HA also function as translational regulatory elements that are controlled by the viral RNA-binding protein (RBP) NS1. The translational increase depends on the nucleotide composition and 5' positioning of the ER-targeting sequence coding regions and is facilitated by the RNA-binding domain of NS1, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5' UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analyzing human protein mRNA sequences, we found evidence that this mechanism of using 5' coding regions and particular RBPs to achieve gene-specific regulation may extend to human-secreted proteins.

Manipulation of neuraminidase packaging signals and hemagglutinin residues improves the growth of A/Anhui/1/2013 (H7N9) influenza vaccine virus yield in eggs.

In 2013, a novel avian-origin H7N9 influenza A virus causing severe lower respiratory tract disease in humans emerged in China, with continued sporadic cases. An effective vaccine is needed for this virus in case it acquires transmissibility among humans; however, PR8-based A/Anhui/1/2013 (Anhui/1, H7N9), a WHO-recommended H7N9 candidate vaccine virus (CVV) for vaccine production, does not replicate well in chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we explored the possibility that PR8's hemagglutinin (HA) and neuraminidase (NA) packaging signals mediate improvement of Anhui/1 CVV yield in eggs. We constructed chimeric HA and NA genes having the coding region of Anhui/1 HA and NA flanked by the 5' and 3' packaging signals of PR8's HA and NA, respectively. The growth of CVVs containing the chimeric HA was not affected, but that of those containing the chimeric NA gene grew in embryonated chicken eggs with a more than 2-fold higher titer than that of WT CVV. Upon 6 passages in eggs further yield increase was achieved although this was not associated with any changes in the chimeric NA gene. The HA of the passaged CVV, did, however, exhibit egg-adaptive mutations and one of them (HA-G218E) improved CVV growth in eggs without significantly changing antigenicity. The HA-G218E substitution and a chimeric NA, thus, combine to provide an Anhui/1 CVV with properties more favorable for vaccine manufacture.

Quantitative analysis of the yield of avian H7 influenza virus haemagglutinin protein produced in silkworm pupae with the use of the codon-optimized DNA: A possible oral vaccine.

In this study, we aimed to quantitatively compare the increased production of three H7 influenza virus-like particle (VLP) haemagglutinin (HA) with the use of a codon-optimized single HA gene in silkworm pupae. Recombinant baculovirus (Korea H7-BmNPV) could produce 0.40 million HA units per pupa, corresponding to 1832µg protein. The yield of the HA produced in larva was estimated to be approximately 0.31 million HA units per larva, and there were no significant differences between the three HA proteins. We could establish efficient recovery system of HA production in larvae and pupae with the use of three cycles sonication methods. Next, we compared yields of HA proteins from three different H7 and two H5 recombinant baculoviruses based on the amount of mRNA synthesized in BmN cells, suggesting that mRNA synthesis may be also a useful indicator for the production of HA. Based on HA titres from four recombinants, the yield of HA had a great influence on the codon-optimized effect and the characteristics of the viral HA gene. The recombinant containing codon optimized HA DNA of A/tufted duck/Fukushima/16/2011 (H5N1) did produce more than one million HA units, although another recombinant including of the wild H5N1 strain failed to show HA activity. Electron microscopy revealed the presence of large VLP and small HA particle in the heavy and light fractions. The purified VLPs reacted with the authentic anti-H7 antibodies and the antibodies prepared after immunization with the VLP H7 antigen. Also H5 and H7VLPs could produce HI antibody in chickens and mice with oral immunization. The antibodies elicited with oral immunization were confirmed in fluorescent antibody analysis and western blotting in Korea H5-BmNPV and H7HA-BmNPV recombinant infected BmN cells. Taken together, these findings provided important insights into future oral vaccine development.

Recombinant production of influenza hemagglutinin and HIV-1 GP120 antigenic peptides using a cleavable self-aggregating tag.

The increasing demand for antigenic peptides in the development of novel serologic diagnostics and epitope-based vaccines requires rapid and reliable peptide synthesis techniques. Here we investigated a method for efficient recombinant expression and purification of medium- to large-sized antigenic peptides in E. coli. Previously we devised a streamlined protein expression and purification scheme based on a cleavable self-aggregating tag (cSAT), which comprised an intein molecule and a self-aggregating peptide ELK16. In this scheme, the target proteins were fused in the C-termini with cSAT and expressed as insoluble aggregates. After intein self-cleavage, target proteins were released into the soluble fraction with high yield and reasonable purity. We demonstrated the applicability of this scheme by preparing seven model viral peptides, with lengths ranging from 32 aa to 72 aa. By adding an N-terminal thioredoxin tag, we enhanced the yield of target peptides released from the aggregates. The purified viral peptides demonstrated high antigenic activities in ELISA and were successfully applied to dissecting the antigenic regions of influenza hemagglutinin. The cSAT scheme described here allows for the rapid and low-cost preparation of multiple antigenic peptides for immunological screening of a broad range of viral antigens.

Production of Influenza Virus HA1 Harboring Native-Like Epitopes by Pichia pastoris.

The outbreak of the H5N1 highly pathogenic avian influenza which exhibits high variation had brought a serious threat to the safety of humanity. To overcome this high variation, hemagglutinin-based recombinant subunit vaccine with rational design has been considered as a substitute for traditional virion-based vaccine development. Here, we expressed HA1 part of the hemagglutinin protein using the Pichia pastoris expression system and attained a high yield of about 120 mg/L through the use of fed-batch scalable fermentation. HA1 protein in the culture supernatant was purified using two-step ion-exchange chromatography. The resultant HA1 protein was homogeneous in solution in a glycosylated form, as confirmed by endoglycosidase H treatment. Sedimentation velocity tests, silver staining of protein gels, and immunoblotting were used for verification. The native HA1 reacted well with conformational, cross-genotype, neutralizing monoclonal antibodies, whereas a loss of binding activity was noted with the denatured HA1 form. Moreover, the murine anti-HA1 serum exhibited a virus-capture capability in the hemagglutination inhibition assay, which suggests that HA1 harbors native-like epitopes. In conclusion, soluble HA1 was efficiently expressed and purified in this study. The functional glycosylated protein will be an alternative for the development of recombinant protein-based influenza vaccine.

Influenza A Viruses Expressing Intra- or Intergroup Chimeric Hemagglutinins.

A panel of influenza A viruses expressing chimeric hemagglutinins (cHA) with intragroup or intergroup head/stalk combinations was generated. Viruses were characterized for growth kinetics and preservation of stalk epitopes. With a few notable exceptions, cHA viruses behaved similarly to wild-type viruses and maintained stalk epitopes, which indicated their potential as vaccine candidates to induce stalk-specific antibodies.

Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology.

UNLABELLED: The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like. IMPORTANCE: Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two- and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

Influenza virus activating host proteases: Identification, localization and inhibitors as potential therapeutics.

Cellular proteases are reponsible for activation of influenza virus hemagglutinin (HA) in epithelial tissues of the respiratory tract. The trans-Golgi network (TGN) is the main subcellular compartment where HA cleavage occurs during its biosynthesis. The proteolytic HA cleavage is an indispensable prerequisite for the fusion of viral with endosomal membrane and the delivery of the virus genome into the cell. Both, the structure and accessibility of the HA cleavage site determine the responsible host protease(s) for cutting. Most influenza virus strains contain a HA sequence with a single arginine at the cleavage site suitable for processing by the trypsin-like serine proteases human airway trypsin-like protease (HAT) and transmembrane protease serine 2 (TMPRSS2), albeit a minority of viruses possesses HA cleavage site motifs that are processed by other proteases. TMPRSS2-deficient mice demonstrated the relevance of TMPRSS2 for pneumotropism and pathogenicity of H1N1 and H7N9 virus infections. In contrast, H3N2 virus infections are promoted by an additional not yet identified protease. Highly pathogenic avian H5 and H7 viruses are characterized by an enlarged cleavage site loop containing a multibasic amino acid motif, where the eukaryotic subtilases furin or PC5/6 cleave. Their ubiquitous presence in the organism allows a systemic virus infection. Peptidomimetic inhibitors derived from the HA cleavage site inhibit the HA-activating proteases and thus virus propagation.

Expression of avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) under control of the ptcB promoter in Lactococcus lactis.

Gram-positive and nonpathogenic lactic acid bacteria (LAB) are considered to be promising candidates for the development of new, safe systems of heterologous protein expression. Recombinant LAB has been shown to induce specific local and systemic immune response against selected pathogens, and could be a good alternative to classical attenuated carriers. The main goal of our study was to express the avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) in Lactococcus lactis. Results of this study were anticipated to lead to construction of lactococcal strain(s) with potential vaccine properties against the avian influenza A (H5N1) virus. Expression of the cloned H5 gene, its His-tagged variant and chIL-2 gene, under the control of the ptcB gene promoter was attested by RT-PCR on transcriptional level and Western or dot blot analysis on translational level, demonstrating that system can be an attractive solution for production of heterologous proteins. The results of the preliminary animal trial conducted in mice are a promising step toward development of a vaccine against avian bird flu using Lactococcus lactis cells as antigen carriers.

Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.

The A/swan/Poland/305-135V08/2006 (H5N1-subtype) hemagglutinin (HA) gene was cloned and expressed in yeast Pichia pastoris (P. pastoris). The HA cDNA lacking the C-terminal transmembrane anchor-coding sequence was fused to an α-factor leader peptide and placed under control of the methanol-inducible P. pastoris alcohol oxidase 1 (AOX1) promoter. Two P. pastoris strains: SMD 1168 and KM 71 were used for protein expression. Recombinant HA protein was secreted into the culture medium reaching an approximately 15 mg/L (KM 71 strain). Fusion protein with a His6 tag was purified to homogeneity in one step affinity chromatography. SDS-PAGE and MS/MS analysis indicated that the protein is cleaved into HA1 and HA2 domains linked by a disulfide bond. Analysis of the N-linked glycans revealed that the overexpressed HA is fully glycosylated at the same sites as the native HA in the vaccine strain. Immunological activity of the hemagglutinin protein was tested in mice, where rHA elicited a high immune response.

Influenza virus hemagglutinin as a vaccine antigen produced in bacteria.

Recombinant subunit vaccines based on hemagglutinin proteins produced in bacteria (bacterial HAs) are promising candidates for enhancing the supply of vaccines against influenza, especially for a pandemic. Over 20 years after the failure to obtain the antigen with native HA characteristics in the early 1980's, there are increasing data on successful production of HA proteins in bacteria. The vast majority of bacterial HAs have been based on the HA1 subunit of HA expressed separately or as a component of conjugate vaccines, but those based on the ectodomain and the HA2 subunit have also been reported. The most of HAs have been efficiently expressed as insoluble aggregates called inclusion bodies. Refolded and purified proteins were extensively studied for structure, the ability to bind to sialic acid-containing receptors, antigenicity, immunogenicity and efficacy. The results from these studies contradict the view that glycosylation determines the correct structure of the hemagglutinin, as they proved that bacterial HAs can be valuable vaccine antigens when appropriate folding and purification methods are applied to rationally designed proteins. The best evidence for success in bacterial production of protective HA is that vaccines based on proprietary Toll-like Receptor (VaxInnate) and bacteriophage Qß-VLPs (Cytos Biotechnology) technologies have been advanced to clinical studies.

Plant expression systems for production of hemagglutinin as a vaccine against influenza virus.

Many examples of a successful application of plant-based expression systems for production of biologically active recombinant proteins exist in the literature. These systems can function as inexpensive platforms for the large scale production of recombinant pharmaceuticals or subunit vaccines. Hemagglutinin (HA) is a major surface antigen of the influenza virus, thus it is in the centre of interests of various subunit vaccine engineering programs. Large scale production of recombinant HA in traditional expression systems, such as mammalian or insect cells, besides other limitations, is expensive and time-consuming. These difficulties stimulate an ever-increasing interest in plant-based production of this recombinant protein. Over the last few years many successful cases of HA production in plants, using both transient and stable expression systems have been reported. Various forms of recombinant HA, including monomers, trimers, virus like particles (VLPs) or chimeric proteins containing its fusion with other polypeptides were obtained and shown to maintain a proper antigenicity. Immunizations of animals (mice, ferrets, rabbits or chickens) with some of these plant-derived hemagglutinin variants were performed, and their effectiveness in induction of immunological response and protection against lethal challenge with influenza virus demonstrated. Plant-produced recombinant subunit vaccines and plant-made VLPs were successfully tested in clinical trials (Phase I and II) that confirmed their tolerance and immunogenicity.