Selective Capture and Determination of Receptor-Binding Hemagglutinin in Influenza Vaccine Preparations Using a Coupled Receptor-Binding/RP-HPLC Assay.

Influenza vaccine potency is determined by the quantification of immunologically active hemagglutinin capable of eliciting neutralizing antibodies upon immunization. Currently, the single radial immunodiffusion (SRID) method is the standard in vitro potency assay used for lot release of seasonal inactivated influenza vaccines. Despite the proven usage of SRID, significant limitations such as the time-consuming preparation of reagents and limited dynamic range warrant the need for the development of alternative potency assays. Such alternative approaches need to discriminate and quantify relevant hemagglutinin material, provide strain identity, and be independent of strain-specific and seasonal reagents. Herein, we present a proof of concept method that combines the capture of conformationally well-folded hemagglutinin via a sialic acid binding step with the resolving power of reversed-phase high-performance liquid chromatography for strain identity and determination. Details of the protocol for the selective capture of receptor-binding hemagglutinin, its release from the receptor, and its relative determination are presented. This approach was found to provide flexibility for the reagents to be used and was adaptable to varying strain compositions of influenza vaccines. This proof of concept approach was developed as an antibody-independent methodology.

The universal epitope of influenza A viral neuraminidase fundamentally contributes to enzyme activity and viral replication.

The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.

Surface modifications of influenza proteins upon virus inactivation by ß-propiolactone.

Inactivation of intact influenza viruses using formaldehyde or ß-propiolactone (BPL) is essential for vaccine production and safety. The extent of chemical modifications of such reagents on viral proteins needs to be extensively investigated to better control the reactions and quality of vaccines. We have evaluated the effect of BPL inactivation on two candidate re-assortant vaccines (NIBRG-121xp and NYMC-X181A) derived from A/California/07/2009 pandemic influenza viruses using high-resolution FT-ICR MS-based proteomic approaches. We report here an ultra performance LC MS/MS method for determining full-length protein sequences of hemagglutinin and neuraminidase through protein delipidation, various enzymatic digestions, and subsequent mass spectrometric analyses of the proteolytic peptides. We also demonstrate the ability to reliably identify hundreds of unique sites modified by propiolactone on the surface of glycoprotein antigens. The location of these modifications correlated with changes to protein folding, conformation, and stability, but demonstrated no effect on protein disulfide linkages. In some cases, these modifications resulted in suppression of protein function, an effect that correlated with the degree of change of the modified amino acids' side chain length and polarity.

A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains.

All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222-230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus.

Synthetic vaccine affords full protection to mice against lethal challenge of influenza B virus of both genetic lineages.

A quarter of all seasonal influenza cases are caused by type B influenza virus (IBV) that also dominates periodically. Here, we investigated a recombinant adenovirus vaccine carrying a synthetic HA2 representing the consensus sequence of all IBV hemagglutinins. The vaccine fully protected mice from lethal challenges by IBV of both genetic lineages, demonstrating its breadth of protection. The protection was not mediated by neutralizing antibodies but robust antibody-dependent cellular cytotoxicity and cell-mediated immune responses. Complete protection of the animals required the entire codon-optimized HA2 sequence that elicited a balanced immune response, whereas truncated vaccines without either the fusion peptide or the transmembrane domain reduced the efficacy of protection. Finally, the vaccines did not demonstrate any sign of disease exacerbation following lung pathology and morbidity monitoring. Collectively, these data suggest that it could be worth further exploring this prototype universal vaccine because of its considerable efficacy, safety, and breadth of protection.

Dysregulation of Ephrin receptor and PPAR signaling pathways in neural progenitor cells infected by Zika virus.

Zika virus (ZIKV) infection is a serious public threat with cases reported in about 70 countries and territories. One of the most serious consequences of ZIKV infection is congenital microcephaly in babies. Congenital microcephaly has been suggested to result from infection of neural progenitor cells (NPCs) in the developing fetal brain. However, the molecular and cellular mechanisms underlying microcephaly development remains to be fully elucidated. In this study, we employed quantitative proteomics to determine protein expression profile that occur during viral replication in NPCs. Bioinformatics analysis of the protein expression changes resulted in the identification of a wide range of cell signaling pathways. Specifically, pathways involved in neurogenesis and embryonic development were markedly altered, along with those associated with cell cycle, apoptosis, lipid metabolism and oxidative stress. Notably, the differential regulation of Ephrin Receptor and PPAR signaling pathways, as revealed by quantitative proteomics and validated by qPCR array, underscores the need to explore these pathways in disease development. Collectively, these results indicate that ZIKV-induced pathogenesis involves complex virus-host reactions; the findings reported here could help shed light on the mechanisms underlying ZIKV-induced microcephaly and ZIKV replication in NPCs.

Unveiling Integrated Functional Pathways Leading to Enhanced Respiratory Disease Associated With Inactivated Respiratory Syncytial Viral Vaccine.

Respiratory syncytial virus (RSV) infection is a severe threat to young children and the elderly. Despite decades of research, no vaccine has been approved. Notably, instead of affording protection, a formalin-inactivated RSV vaccine induced severe respiratory disease including deaths in vaccinated children in a 1960s clinical trial; however, recent studies indicate that other forms of experimental vaccines can also induce pulmonary pathology in pre-clinical studies. These findings suggest that multiple factors/pathways could be involved in the development of enhanced respiratory diseases. Clearly, a better understanding of the mechanisms underlying such adverse reactions is critically important for the development of safe and efficacious vaccines against RSV infection, given the exponential growth of RSV vaccine clinical trials in recent years. By employing an integrated systems biology approach in a pre-clinical cotton rat model, we unraveled a complex network of pulmonary canonical pathways leading to disease development in vaccinated animals upon subsequent RSV infections. Cytokines including IL-1, IL-6 GRO/IL-8, and IL-17 in conjunction with mobilized pulmonary inflammatory cells could play important roles in disease development, which involved a wide range of host responses including exacerbated pulmonary inflammation, oxidative stress, hyperreactivity, and homeostatic imbalance between coagulation and fibrinolysis. Moreover, the observed elevated levels of MyD88 implicate the involvement of this critical signal transduction module as the central node of the inflammatory pathways leading to exacerbated pulmonary pathology. Finally, the immunopathological consequences of inactivated vaccine immunization and subsequent RSV exposure were further substantiated by histological analyses of these key proteins along with inflammatory cytokines, while hypercoagulation was supported by increased pulmonary fibrinogen/fibrin accompanied by reduced levels of plasma D-dimers. Enhanced respiratory disease associated with inactivated RSV vaccine involves a complex network of host responses, resulting in significant pulmonary lesions and clinical manifestations such as tachypnea and airway obstruction. The mechanistic insight into the convergence of different signal pathways and identification of biomarkers could help facilitate the development of safe and effective RSV vaccine and formulation of new targeted interventions.

Targeting CD40 enhances antibody- and CD8-mediated protection against respiratory syncytial virus infection.

Respiratory Syncytial Virus (RSV) infects almost all children under the age of one and is the leading cause of hospitalization among infants. Despite several decades of research with dozens of candidate vaccines being vigorously evaluated in pre-clinical and clinical studies, there is no licensed vaccine available to date. Here, the RSV fusion protein (F) was fused with CD40 ligand and delivered by an adenoviral vector into BALB/c mice where the CD40 ligand serves two vital functions as a molecular adjuvant and an antigen-targeting molecule. In contrast to a formaldehyde-inactivated vaccine, the vectored vaccine effectively protected animals against RSV without inducing enhanced respiratory disease. This protection involved a robust induction of neutralizing antibodies and memory CD8 T cells, which were not observed in the inactivated vaccine group. Finally, the vectored vaccine was able to elicit long-lasting protection against RSV, one of the most challenging issues in RSV vaccine development. Further studies indicate that the long lasting protection elicited by the CD40 ligand targeted vaccine was mediated by increased levels of effector memory CD8 T cell 3 months post-vaccination.

Topological N-glycosylation and site-specific N-glycan sulfation of influenza proteins in the highly expressed H1N1 candidate vaccines.

The outbreak of a pandemic influenza H1N1 in 2009 required the rapid generation of high-yielding vaccines against the A/California/7/2009 virus, which were achieved by either addition or deletion of a glycosylation site in the influenza proteins hemagglutinin and neuraminidase. In this report, we have systematically evaluated the glycan composition, structural distribution and topology of glycosylation for two high-yield candidate reassortant vaccines (NIBRG-121xp and NYMC-X181A) by combining various enzymatic digestions with high performance liquid chromatography and multiple-stage mass spectrometry. Proteomic data analyses of the full-length protein sequences determined 9 N-glycosylation sites of hemagglutinin, and defined 6 N-glycosylation sites and the glycan structures of low abundance neuraminidase, which were occupied by high-mannose, hybrid and complex-type N-glycans. A total of ~300 glycopeptides were analyzed and manually validated by tandem mass spectrometry. The specific N-glycan structure and topological location of these N-glycans are highly correlated to the spatial protein structure and the residential ligand binding. Interestingly, sulfation, fucosylation and bisecting N-acetylglucosamine of N-glycans were also reliably identified at the specific glycosylation sites of the two influenza proteins that may serve a crucial role in regulating the protein structure and increasing the protein abundance of the influenza virus reassortants.

The molecular determinants governing the immunogenicity of Japanese encephalitis live attenuated vaccines.

In the course of isolating the attenuated Japanese encephalitis vaccine SA14-14-2, two attenuated strains SA14-9-7 and SA14-5-3 were also obtained that elicited low antibody responses in humans (<10% and 62%, respectively) and exerted much weaker immune protection in animal challenge experiments. However, the reason for these differences remains unknown. In order to understand why SA14-14-2 is superior to SA14-9-7 and SA14-5-3, we employed a reverse genetics method to identify the key mutations in the virus genome that determine the immunogenicity of live attenuated Japanese encephalitis viruses. We first sequenced the full genomic sequences of SA14-9-7 and SA14-5-3 and found mutations that changed four amino-acid base pairs when compared to the envelope gene of SA14-14-2. We mutated the genome of SA14-14-2 to generate these mutations both singly (E-177, E-264, E-279 and E-315) and in combination (E-177/264, E-279/315 and E-177/264/279/315) and tested these mutants along with parental strains SA14-14-2, SA14-9-7 and SA14-5-3 for their immunogenicity in vivo. When mice were immunized with SA14-9-7 and SA14-5-3, lower levels of neutralizing antibodies were generated compared with the immune response to SA14-14-2. Furthermore, SA14-5-3 was more immunogenic than SA14-9-7, which replicated the results previously seen in humans. Point mutations E-177, E-264, E-279 and E-315 diminished the immunogenicity of SA14-14-2 with E-264 and E-315, contributing the most to this phenotype. The mutant rJEV (E-177/E-264/E-279/E-315) containing all four point mutations exhibited the lowest immunogenicity with a seroconversion rate of 0 at an inoculation dose of 103 PFU (plaque-forming unit). We have identified the key amino acids in the envelope protein that account for the superior immunogenicity of SA14-14-2.

Development and applications of universal H7 subtype-specific antibodies for the analysis of influenza H7N9 vaccines.

H7N9 is a newly emerged avian influenza virus with a relatively high mortality rate in humans. At this time, there is no licensed vaccine for human protection. Development of analytical tools for H7N9 vaccine could facilitate vaccine development. Here, a universally conserved epitope in all H7 hemagglutinin (HA) sequences was identified through comprehensive bioinformatics analyses. The peptide epitope, RSGSSFYAEMK, (aa positions 149 to 159), is located on the head of the HA molecule. Antibodies generated against this universal H7 epitope were remarkably specific against H7 viral sequence with no detectable cross-reactivity to other HA subtypes. A new immunoblotting assay based on the universal H7 antibody was developed and compared with the traditional single radial immunodiffusion assay (SRID) for potency analyses of candidate H7N9 vaccines. This new assay was more sensitive and rapid compared to SRID. In addition to statistically acceptable precision and reproducibility, the new assay differs from many other alternative potency assays for influenza vaccine in that it is potentially stability-indicating, which is an important requirement for industry vaccine stability studies analyses. Furthermore, the robustness of this new assay was demonstrated by the quantitative determination of HA content in four H7N9 vaccines (split or inactivated) from different manufacturers.

A novel synthetic receptor-based immunoassay for influenza vaccine quantification.

Vaccination is the most effective prophylactic method for preventing influenza. Quantification of influenza vaccine antigens is critically important before the vaccine is used for human immunization. Currently the vaccine antigen quantification relies on hemagglutinin content quantification, the key antigenic component, by single radial immunodiffusion (SRID) assay. Due to the inherent disadvantages associated with the traditional SRID; i.e. low sensitivity, low throughput and need for annual reagents, several approaches have been proposed and investigated as alternatives. Yet, most alternative methods cannot distinguish native hemagglutinin from denatured form, making them less relevant to antigenic analyses. Here, we developed a quantitative immunoassay based on the sialic acid binding property of influenza vaccine antigens. Specifically, we chemically synthesized human and avian influenza virus receptors analogues, N-acetylneuraminic acid-2,6-lactose and N-acetylneuraminic acid-2,3-lactose derivatives with an azidopropyl aglycon, using α-2,6- and α-2,3-sialyltransferases, respectively. The azido group of the two sialyllactose-derivatives was reduced and conjugated to mouse serum albumin through a squarate linkage. We showed that the synthetic α-2,6- and α-2,3-receptors selectively bound to human and avian-derived hemagglutinins, respectively, forming the basis of a new, and robust assay for hemagglutinin quantification. Hemagglutinin treated at high temperature or low pH was measured differentially to untreated samples suggesting native conformation is dependent for optimal binding. Importantly, this receptor-based immunoassay showed excellent specificity and reproducibility, high precision, less turnaround time and significantly higher sensitivity and throughput compared with SRID in analyzing multiple influenza vaccines.

Universal anti-neuraminidase antibody inhibiting all influenza A subtypes.

The only universally conserved sequence amongst all influenza A viral neuraminidase (NA) is located between amino acids 222-230 and plays crucial roles in viral replication. However, it remained unclear as to whether this universal epitope is exposed during the course of infection to allow binding and inhibition by antibodies. Using a monoclonal antibody (MAb) targeting this specific epitope, we demonstrated that all nine subtypes of NA were inhibited in vitro by the MAb. Moreover, the antibody also provided heterosubtypic protection in mice challenged with lethal doses of mouse-adapted H1N1 and H3N2, which represent group I and II viruses, respectively. Furthermore, we report amino acid residues I222 and E227, located in close proximity to the active site, are indispensable for inhibition by this antibody. This unique, highly-conserved linear sequence in viral NA could be an attractive immunological target for protection against diverse strains of influenza viruses.

Modifying the thermostability of inactivated influenza vaccines.

BACKGROUND: Respiratory infections caused by influenza viruses spread rapidly, resulting in significant annual morbidity and mortality worldwide. Currently, the most effective public health measure against infection is immunisation with an influenza vaccine matching the relevant circulating influenza strains. Although a number of developments in terms of influenza vaccine production, safety and immunogenicity have been reported, limitations in our understanding of vaccine stability still exist. In this report we seek to identify compounds that increase influenza vaccine thermostability. METHODS: We use plaque inhibition on confluent MDCK cells to identify compounds which inhibit the entry of various seed strain viruses. The effect of these compounds on vaccine thermal lability is evaluated through SRID analysis. The significance of these results is tested by a two-way analysis of variance (ANOVA) method. RESULTS: We identify two compounds which selectively inhibit entry of different group I or group II influenza strains through prevention of the neutral-pH to low-pH conformational change of hemagglutinin. Compounds which were able to inhibit virus entry were also able to limit thermally induced potency loss in matched influenza vaccines. Furthermore, we demonstrate that this effect is independent of product formulation or the presence of multiple HA types. CONCLUSIONS: This work provides further evidence for a link between HA conformational stability in the virus and thermostability of the corresponding vaccine preparation. It also suggests straightforward approaches to improve the stability and predictability of influenza vaccine preparations.

Simultaneous quantification of the viral antigens hemagglutinin and neuraminidase in influenza vaccines by LC-MSE.

Current methods for quality control of inactivated influenza vaccines prior to regulatory approval include determining the hemagglutinin (HA) content by single radial immunodiffusion (SRID), verifying neuraminidase (NA) enzymatic activity, and demonstrating that the levels of the contaminant protein ovalbumin are below a set threshold of 1 µg/dose. The SRID assays require the availability of strain-specific reference HA antigens and antibodies, the production of which is a potential rate-limiting step in vaccine development and release, particularly during a pandemic. Immune responses induced by neuraminidase also contribute to protection from infection; however, the amounts of NA antigen in influenza vaccines are currently not quantified or standardized. Here, we report a method for vaccine analysis that yields simultaneous quantification of HA and NA levels much more rapidly than conventional HA quantification techniques, while providing additional valuable information on the total protein content. Enzymatically digested vaccine proteins were analyzed by LC-MS(E), a mass spectrometric technology that allows absolute quantification of analytes, including the HA and NA antigens, other structural influenza proteins and chicken egg proteins associated with the manufacturing process. This method has potential application for increasing the accuracy of reference antigen standards and for validating label claims for HA content in formulated vaccines. It can also be used to monitor NA and chicken egg protein content in order to monitor manufacturing consistency. While this is a useful methodology with potential for broad application, we also discuss herein some of the inherent limitations of this approach and the care and caution that must be taken in its use as a tool for absolute protein quantification. The variations in HA, NA and chicken egg protein concentrations in the vaccines analyzed in this study are indicative of the challenges associated with the current manufacturing and quality control testing procedures.

Antigenic stability of H1N1 pandemic vaccines correlates with vaccine strain.

In 2009 a novel H1N1 influenza virus emerged and spread rapidly. Soon after vaccine lots were released, however, the shelf life was revised downward due to an unexpected decrease in HA potency. In this study, we found differences in both stability and antigenic content of two monovalent H1N1/2009 vaccine preparations. These appear to have arisen due to differences in the A/California/7/2009-like influenza strain used to prepare vaccine.

Rapid and accurate determination of the potency of varicella vaccine by quantitative polymerase chain reaction.

The potency of varicella vaccines is currently determined by a plaque assay technique, which usually takes seven days and is laborious and has considerable inter- and intra-assay variability. Here, we report a new potency assay for varicella vaccine based on quantitative polymerase chain reaction in conjunction with a much more efficient virus infection step. Potency results can be obtained within 24h of infection and demonstrates acceptable accuracy and reproducibility when compared with the plaque assay, which relies on manual counting of plaques formed one week after viral infection. Using multiple vaccine lots from 7 manufacturers, we found no significant difference in infectivity determined between the new assay and plaque assay. The optimized conditions for viral infection and polymerase chain reaction are of significant value for the potency determination of the vaccine due to its rapidity, accuracy and the high throughput capacity of the assay.

Acetaminophen modulates the transcriptional response to recombinant interferon-beta.

BACKGROUND: Recombinant interferon treatment can result in several common side effects including fever and injection-site pain. Patients are often advised to use acetaminophen or other over-the-counter pain medications as needed. Little is known regarding the transcriptional changes induced by such co-administration. METHODOLOGY/PRINCIPAL FINDINGS: We tested whether the administration of acetaminophen causes a change in the response normally induced by interferon-beta treatment. CD-1 mice were administered acetaminophen (APAP), interferon-beta (IFN-beta) or a combination of IFN-beta+APAP and liver and serum samples were collected for analysis. Differential gene expression was determined using an Agilent 22 k whole mouse genome microarray. Data were analyzed by several methods including Gene Ontology term clustering and Gene Set Enrichment Analysis. We observed a significant change in the transcription profile of hepatic cells when APAP was co-administered with IFN-beta. These transcriptional changes included a marked up-regulation of genes involved in signal transduction and cell differentiation and down-regulation of genes involved in cellular metabolism, trafficking and the IkappaBK/NF-kappaB cascade. Additionally, we observed a large decrease in the expression of several IFN-induced genes including Ifit-3, Isg-15, Oasl1, Zbp1 and predicted gene EG634650 at both early and late time points. CONCLUSIONS/SIGNIFICANCE: A significant change in the transcriptional response was observed following co-administration of IFN-beta+APAP relative to IFN-beta treatment alone. These results suggest that administration of acetaminophen has the potential to modify the efficacy of IFN-beta treatment.

Aurintricarboxylic acid is a potent inhibitor of influenza A and B virus neuraminidases.

BACKGROUND: Influenza viruses cause serious infections that can be prevented or treated using vaccines or antiviral agents, respectively. While vaccines are effective, they have a number of limitations, and influenza strains resistant to currently available anti-influenza drugs are increasingly isolated. This necessitates the exploration of novel anti-influenza therapies. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the potential of aurintricarboxylic acid (ATA), a potent inhibitor of nucleic acid processing enzymes, to protect Madin-Darby canine kidney cells from influenza infection. We found, by neutral red assay, that ATA was protective, and by RT-PCR and ELISA, respectively, confirmed that ATA reduced viral replication and release. Furthermore, while pre-treating cells with ATA failed to inhibit viral replication, pre-incubation of virus with ATA effectively reduced viral titers, suggesting that ATA may elicit its inhibitory effects by directly interacting with the virus. Electron microscopy revealed that ATA induced viral aggregation at the cell surface, prompting us to determine if ATA could inhibit neuraminidase. ATA was found to compromise the activities of virus-derived and recombinant neuraminidase. Moreover, an oseltamivir-resistant H1N1 strain with H274Y was also found to be sensitive to ATA. Finally, we observed additive protective value when infected cells were simultaneously treated with ATA and amantadine hydrochloride, an anti-influenza drug that inhibits M2-ion channels of influenza A virus. CONCLUSIONS/SIGNIFICANCE: Collectively, these data suggest that ATA is a potent anti-influenza agent by directly inhibiting the neuraminidase and could be a more effective antiviral compound when used in combination with amantadine hydrochloride.

Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins.

The fusion peptide is the only universally conserved sequence in the hemagglutinins of all 16 subtypes of influenza A and two genetic lineages of influenza B viruses. Here, peptides selected by bioinformatics approach were modified and conjugated to overcome serious technical hurdles such as the high hydrophobicity and weak immunogenicity of the viral fusion peptides. Antibodies generated against fusion peptides demonstrated remarkable specificity against the viral sequences and robustness of quantitatively analyzing the viral hemagglutinins even under stringent conditions. As quantitatively revealed by antibody-binding experiments, the fusion peptides of diverse hemagglutinins are exposed to the same degree upon unfolding at neutral pH to the physiologically fusogenic state. To our knowledge, this is the first report on the quantitative determination of virtually all influenza vaccines using a single universal antibody.