Identification of immunodominant CD8 epitope in the stalk domain of influenza B viral hemagglutinin.

Human infections by type B influenza virus constitute about 25% of all influenza cases. The viral hemagglutinin is comprised of two subunits, HA1 and HA2. While HA1 is constantly evolving in an unpredictable fashion, the HA2 subunit is highly conserved, making it a potential candidate for a universal vaccine. However, immunodominant epitopes in the HA2 subunit remain largely unknown. To delineate MHC Class I epitopes, we first identified 9-mer H-2Kd-restricted CD8 T cell epitopes in the HA2 domain by in silico analyses, followed by evaluating the immunodominance of these peptides in mice challenged with the virus. Of three peptides selected through in silico analysis, the universally conserved peptide, YYSTAASSL (B/HA2-190), possessed the highest predicted binding affinity to MHC Class I and was most effective in inducing IL-2 and TNF-α in mouse splenocytes. Importantly, the peptide demonstrated best capability of stimulating peptide-specific ex-vivo cytotoxicity against target cells. Taken together, this finding would be of value for assessment of cell-mediated immune responses elicited by vaccines based on the highly conserved HA2 stalk domain.

CD40 ligand preferentially modulates immune response and enhances protection against influenza virus.

CD40L, a key regulator of the immune system, was studied as both a targeting ligand and a molecular adjuvant in nucleoprotein (NP)-based host defense against influenza in mouse models with different genetic backgrounds. Adenoviral vectors secreting NP-CD40L fusion protein (denoted as rAd-SNP40L) afforded full protection of immunocompetent and immunocompromised mice (CD40L(-/-) and CD4(-/-)) against lethal influenza infection. Mechanistically, rAd-SNP40L preferentially induced early and persistent B cell germinal center formation, and accelerated Ig isotype-switching and Th1-skewed, NP-specific Ab response. Moreover, it drastically augmented primary and memory NP-specific CTL activity and polyfunctional CD8(+) T cells. The markedly enhanced nonneutralizing Abs and CTLs significantly reduced viral burdens in the lungs of mice upon lethal virus challenge. Data generated from CD40L(-/-) and CD4(-/-) mice revealed that the protection was indeed CD40L mediated but CD4(+) T cell independent, demonstrating the viability of the fusion Ags in protecting immunodeficient hosts. Notably, a single dose of rAd-SNP40L completely protected mice from lethal viral challenge 4 mo after immunization, representing the first report, to our knowledge, on NP in conjunction with a molecular adjuvant inducing a robust and long-lasting memory immune response against influenza. This platform is characterized by an increased in vivo load of CD40-targeted Ag upon the secretion of the fusion protein from adenovirus-infected cells and may represent a promising strategy to enhance the breadth, durability, and potency of Ag-specific immune responses.

The Expression Kinetics and Immunogenicity of Lipid Nanoparticles Delivering Plasmid DNA and mRNA in Mice.

In recent years, lipid nanoparticles (LNPs) have emerged as a revolutionary technology for vaccine delivery. LNPs serve as an integral component of mRNA vaccines by protecting and transporting the mRNA payload into host cells. Despite their prominence in mRNA vaccines, there remains a notable gap in our understanding of the potential application of LNPs for the delivery of DNA vaccines. In this study, we sought to investigate the suitability of leading LNP formulations for the delivery of plasmid DNA (pDNA). In addition, we aimed to explore key differences in the properties of popular LNP formulations when delivering either mRNA or DNA. To address these questions, we compared three leading LNP formulations encapsulating mRNA- or pDNA-encoding firefly luciferase based on potency, expression kinetics, biodistribution, and immunogenicity. Following intramuscular injection in mice, we determined that RNA-LNPs formulated with either SM-102 or ALC-0315 lipids were the most potent (all p-values < 0.01) and immunogenic (all p-values < 0.05), while DNA-LNPs formulated with SM-102 or ALC-0315 demonstrated the longest duration of signal. Additionally, all LNP formulations were found to induce expression in the liver that was proportional to the signal at the injection site (SM102: r = 0.8787, p < 0.0001; ALC0315: r = 0.9012, p < 0.0001; KC2: r = 0.9343, p < 0.0001). Overall, this study provides important insights into the differences between leading LNP formulations and their applicability to DNA- and RNA-based vaccinations.

Bivalent vaccines effectively protect mice against influenza A and respiratory syncytial viruses.

Influenza and Respiratory Syncytial virus (RSV) infections together contribute significantly to the burden of acute lower respiratory tract infections. Despite the disease burden, no approved RSV vaccine is available. While approved vaccines are available for influenza, seasonal vaccination is required to maintain protection. In addition to both being respiratory viruses, they follow a common seasonality, which warrants the necessity for a concerted vaccination approach. Here, we designed bivalent vaccines by utilizing highly conserved sequences, targeting both influenza A and RSV, as either a chimeric antigen or individual antigens separated by a ribosome skipping sequence. These vaccines were found to be effective in protecting the animals from challenge by either virus, with mechanisms of protection being substantially interrogated in this communication.

Percutaneous Transcatheter Edge-to-Edge Mitral Valve Repair With MitraClip System in the Era of G4.

The use of transcatheter edge-to-edge mitral valve repair (TEER) in symptomatic patients with severe mitral regurgitation (MR) has dramatically increased over the last few years. Current guidelines consider TEER as a reasonable option in symptomatic patients with primary or chronic secondary severe MR with high or prohibitive surgical risk and favorable anatomy. However, several anatomical and morphological mitral features have restricted the use of this mini-invasive technique in its early experience. The latest fourth generation (G4) of the MitraClip system has been recently introduced and includes the possibility of independent leaflet grasping and 4 different sizes. This technical update offers the possibility of selecting and combining multiple devices for complex mitral valve anatomies and challenging procedures, which helps expand the applications of TEER. The present review describes the potential advantages and the help of the MitraClip G4 devices to overcome various anatomic and morphologic issues in challenging cases with complex primary and secondary MR procedures.

DNA lipid nanoparticle vaccine targeting outer surface protein C affords protection against homologous Borrelia burgdorferi needle challenge in mice.

Introduction: The incidence of Lyme disease (LD) in Canada and the United States has risen over the last decade, nearing 480,000 cases each year. Borrelia burgdorferi sensu lato, the causative agent of LD, is transmitted to humans through the bite of an infected tick, resulting in flu-like symptoms and often a characteristic bull's-eye rash. In more severe cases, disseminated bacterial infection can cause arthritis, carditis and neurological impairments. Currently, no vaccine is available for the prevention of LD in humans. Methods: In this study, we developed a lipid nanoparticle (LNP)-encapsulated DNA vaccine encoding outer surface protein C type A (OspC-type A) of B. burgdorferi. Results: Vaccination of C3H/HeN mice with two doses of the candidate vaccine induced significant OspC-type A-specific antibody titres and borreliacidal activity. Analysis of the bacterial burden following needle challenge with B. burgdorferi (OspC-type A) revealed that the candidate vaccine afforded effective protection against homologous infection across a range of susceptible tissues. Notably, vaccinated mice were protected against carditis and lymphadenopathy associated with Lyme borreliosis. Discussion: Overall, the results of this study provide support for the use of a DNA-LNP platform for the development of LD vaccines.

Universal antibody targeting the highly conserved fusion peptide provides cross-protection in mice.

Influenza is a major public health concern causing millions of hospitalizations every year. The current vaccines need annual updating based on prediction of likely strains in the upcoming season. However, mismatches between vaccines and the actual circulating viruses can occur, reducing vaccine effectiveness significantly because of the remarkably high rate of mutation in the viral glycoprotein, hemagglutinin (HA). Clearly, it would be of great interest to determine the potential role of universally conserved epitopes in inducing protective immunity. Here, an antibody against the 14-aa fusion peptide sequence at the N-terminus of the HA2 subunit (Uni-1) was investigated for its ability to elicit antibody-dependent cellular cytotoxicity (ADCC) in vitro and cross-protection against lethal infection in animals. Uni-1, known to neutralize influenza type A (IAV) in vitro, was found to induce strong ADCC against diverse influenza viruses, including human and avian IAVs and both lineages of type B (IBV). The ADCC effects against human IAVs by Uni-1 was comparable to ADCC induced by well-characterized antibodies, F10 and FI6V3. Importantly, mice treated with Uni-1 were protected against lethal challenge of IAV and IBV. These results revealed the versatile effector functions of this universal antibody against markedly diverse strains of both IAV and IBV.

The fusion peptide is the only universally conserved epitope in both IAV and IBVMono-specific universal antibody induces strong ADCC against human and avian IAVMono-specific universal antibody induces strong ADCC against IBV from both genetic lineages of IBVThe antibody has bi-functional effector functions against several influenza viruses.

The effect of interferon-α on the expression of cytochrome P450 3A4 in human hepatoma cells.

Interferon α (IFNα) is used to treat malignancies and chronic viral infections. It has been found to decrease the rate of drug metabolism by acting on cytochrome P450 enzymes, but no studies have investigated the consequences of IFNα treatment on the CYP3A4 isoform, responsible for the metabolism of a majority of drugs. In this study, we have examined the effect of IFNα on CYP3A4 catalytic activity and expression in human hepatoma cells. We found that IFNα inhibits CYP3A4 activity and rapidly down-regulates the expression of CYP3A4, independent of de novo protein synthesis. Pharmacologic inhibitors and a dominant-negative mutant expression plasmid were used to dissect the molecular pathway required for CYP3A4 suppression, revealing roles for Jak1 and Stat1 and eliminating the involvement of the p38 mitogen-activated and extracellular regulated kinases. Treatment of hepatoma cells with IFNα did not affect the nuclear localization or relative abundance of Sp1 and Sp3 transcription factors, suggesting that the suppression of CYP3A4 by IFNα does not result from inhibitory Sp3 out-competing Sp1. To our knowledge, this is the first report that IFNα down-regulates CYP3A4 expression largely through the JAK-STAT pathway. Since IFNα suppresses CYP3A4 expression, caution is warranted when IFNα is administered in combination with CYP3A4 substrates to avoid the occurrence of adverse drug interactions.

Single Immunization of a Vaccine Vectored by a Novel Recombinant Vaccinia Virus Affords Effective Protection Against Respiratory Syncytial Virus Infection in Cotton Rats.

Respiratory syncytial virus (RSV) is a leading cause of respiratory infections worldwide and disease management measures are hampered by the lack of a safe and effective vaccine against the infection. We constructed a novel recombinant RSV vaccine candidate based on a deletion mutant vaccinia virus platform, in that the host range genes E3L and K3L were deleted (designated as VACVΔE3LΔK3L) and a poxvirus K3L ortholog gene was used as a marker for the rapid and efficient selection of recombinant viruses. The safety of the modified vaccinia virus was investigated by intranasal administration of BALB/c mice with the modified vaccinia vector using a dose known to be lethal in the wild-type Western Reserve. Only a minor loss of body weight by less than 5% and mild pulmonary inflammation were observed, both of which were transient in nature following nasal administration of the high-dose modified vaccinia virus. In addition, the viruses were cleared from the lung in 2 days with no viral invasions of the brain and other vital organs. These results suggest that the virulence of the virus has been essentially abolished. We then investigated the efficiency of the vector for the delivery of vaccines against RSV through comparison with another RSV vaccine delivered by the widely used Modified Vaccinia virus Ankara (MVA) backbone. In the cotton rats, we found a single intramuscular administration of VACVΔE3LΔK3L-vectored vaccine elicited immune responses and protection at a level comparable to the MVA-vectored vaccine against RSV infection. The distinct features of this novel VACV vector, such as an E3L deletion for attenuation and a K3L ortholog for positive selection and high efficiency for vaccine delivery, could provide unique advantages to the application of VACV as a platform for vaccine development.

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.

DNA Based Vaccine Expressing SARS-CoV-2 Spike-CD40L Fusion Protein Confers Protection Against Challenge in a Syrian Hamster Model.

SARS-CoV-2 infections present a tremendous threat to public health. Safe and efficacious vaccines are the most effective means in preventing the infections. A variety of vaccines have demonstrated excellent efficacy and safety around the globe. Yet, development of alternative forms of vaccines remains beneficial, particularly those with simpler production processes, less stringent storage conditions, and the capability of being used in heterologous prime/boost regimens which have shown improved efficacy against many diseases. Here we reported a novel DNA vaccine comprised of the SARS-CoV-2 spike protein fused with CD40 ligand (CD40L) serving as both a targeting ligand and molecular adjuvant. A single intramuscular injection in Syrian hamsters induced significant neutralizing antibodies 3-weeks after vaccination, with a boost substantially improving immune responses. Moreover, the vaccine also reduced weight loss and suppressed viral replication in the lungs and nasal turbinates of challenged animals. Finally, the incorporation of CD40L into the DNA vaccine was shown to reduce lung pathology more effectively than the DNA vaccine devoid of CD40L. These results collectively indicate that this DNA vaccine candidate could be further explored because of its efficacy and known safety profile.

Characteristics and Outcomes of Patients Who Are Denied From a Percutaneous Edge-to-Edge Mitral Valve Repair After Being Referred to a Transcatheter Mitral Valve Program: Impact of a Dedicated Multidisciplinary Mitral Heart Team Approach.

BACKGROUND: Many patients referred for a MitraClip intervention are finally refused for this intervention, and data are very scarce on their outcomes. Our study sought to determine the characteristics and outcomes of patients who are referred to a mitral valve clinic and are finally denied from a percutaneous mitral edge-to-edge repair. METHODS: A total of 210 patients referred to our clinic for severe mitral regurgitation were retrospectively analyzed. Fifty-seven patients underwent a MitraClip procedure. For exploratory purposes, a propensity-matched cohort comparing the patients accepted for a MitraClip procedure and those refused for any mitral intervention was analyzed. RESULTS: Among the 153 patients who were refused for MitraClip, 46% had functional MR, 42% had degenerative MR, and 11% had mixed disease. Reasons for denial included unfavorable anatomy, patient refusal, mitral valve surgery referral, cardiac resynchronization therapy, other advanced heart failure therapies, and palliative care. After a mean follow-up of 13 months, 50% were in New York Heart Association class I or II, 63% had less than severe MR, and mortality rate was 29%. In the propensity-matched cohort, there was no difference in symptoms improvement, but there was less overall mortality (P=.01), cardiovascular mortality (P<.01) and severe MR (P<.01) in the MitraClip group. CONCLUSIONS: A multidisciplinary heart team evaluation for complex MR patients can be useful not solely for selecting the ideal MitraClip eligible patients, but also to select the best treatment strategy in each individualized context.

Chitosan alters inactivated respiratory syncytial virus vaccine elicited immune responses without affecting lung histopathology in mice.

Chitosan is a polysaccharide capable of augmenting immune responses with a proven safety record in animals and humans. These properties make it a potentially attractive agent for the prevention and treatment of infectious disease. Infection by respiratory syncytial virus (RSV) is the leading cause of serious lower respiratory disease in young children throughout the world. There is no licensed vaccine available against RSV whereas inactivated vaccine is known to cause enhanced respiratory disease instead of protection. Here, we investigated whether chitosan administered one or three days post-infection could protect animals against RSV infection and whether it could alter immune responses or immunopathology induced by inactivated RSV vaccine when administered twice before RSV infection. We found chitosan could modestly protect animals against RSV infection when given post-infection, while, in conjunction with inactivated RSV vaccine when given pre-infection, it could significantly reduce RSV infection in mice. Further mechanistic investigation revealed that chitosan enhanced antigen-specific immune responses through augmenting the induction of regulatory T cells, lung resident T cells and neutralizing antibodies while reversing Th2-skewed immune responses induced by inactivated RSV vaccine but, surprisingly, failing to reverse lung histopathology. Overall, this study sheds more light on the molecular mechanisms underlying inactivated RSV vaccine-induced disease.

Universal type/subtype-specific antibodies for quantitative analyses of neuraminidase in trivalent influenza vaccines.

Both influenza viral hemagglutinin and neuraminidase can induce protective immune responses in humans. Although the viral hemagglutinin antigens have been quantified in influenza vaccines, the amounts of neuraminidase remain undetermined. Using comprehensive bioinformatics analyses of all neuraminidase sequences, we identified highly conserved and subtype-specific peptide epitopes within each of N1, N2 and type B neuraminidase groups. Mono-specific antibodies generated against these peptides bound to their respective subtype/type only while demonstrating remarkable specificity against the viral neuraminidase sequences without any cross-reactivity with allantoic and cellular proteins. Moreover, the subtype/type-specific antibodies were found not to interfere with one another when a mixture of vaccine samples was analysed. Importantly, immunoassay based on these antibodies can quantitatively determine neuraminidase in commercial trivalent vaccine samples. Analyses of vaccines from eight manufacturers using the same vaccine seeds revealed significant differences in neuraminidase levels. Specifically, while the ratio between neuraminidase and hemagglutinin in some products are found to be close 1/5, other products have a ratio of approximately 1/100, a level which is far below the theoretical ratio between neuraminidase and hemagglutinin in a virus. The antibody-based assays reported here could be of great value for better quality control of both monovalent and trivalent vaccines.

A voluntary conservation agreement reduces the risks of lethal collisions between ships and whales in the St. Lawrence Estuary (Québec, Canada): From co-construction to monitoring compliance and assessing effectiveness.

Lethal collisions with ships are limiting the recovery of several at-risk whale species worldwide. In the St. Lawrence Estuary (Quebec, Canada), the endangered blue whale and of special concern fin whale are among the migratory species subject to collisions with large ships. In 2011, a working group composed of representatives from the maritime industry, the government, non-governmental organizations, and academia was created to explore solutions to mitigate ship-whale collisions in the St. Lawrence Estuary. Adopting an adaptive risk management framework, the working group took advantage of the best available scientific data and tools to co-construct realistic collision mitigation options and evaluate their likely benefits for whale conservation and costs for the industry. In 2013, the working group recommended the implementation of voluntary measures to mitigate collision risks, consisting of a slow-down area, a no-go area, and a caution area; a recommended route was added in 2014. Along with the voluntary framework, the working group agreed to continuously monitor compliance with and assess effectiveness of these mitigation measures. After the fourth year of implementation, voluntary measures showed encouraging results, with a reduction of up to 40% of lethal collision risks with fin whales in the highest density area. This reduction in risk is mainly related to ship speed reduction in the slow-down area from 14.1 ± 2.6 knots in 2012 to 11.3 ± 1.7 knots since 2014. The presence of a mandatory pilotage area overlapping with the slow-down area was instrumental to facilitate communication about the mitigation measures, with the pilotage corporation sitting as a regular member of the working group. This resulted in significantly slower speeds in the slow-down area for ships with a pilot from the pilotage corporation onboard compared to those without (-0.8 knots, p-value < 0.001). It is also likely to explain the weaker compliance of the maritime industry with the no-go area located outside of the mandatory pilotage area. Other factors of success include: the continuous dedication of the government to a voluntary and transparent participatory process; the use of available data, tools and institutions; the presence of an environmental certification program representative in the working group; and the adoption by consensus of an adaptive risk management approach. The traditional regulatory approach to conservation is often blamed for its focus on deterring negative behaviors, doing nothing to encourage and reward positive ones. In agreement with other case studies, the benefits of the voluntary measures implemented in the St. Lawrence Estuary include the pro-active commitment from the industry (which is likely to reduce conflicts with regulators), the greater flexibility and freedom that allowed to come up with cost-effective and tailored-made mitigation measures, and the fast achievement of conservation gains. More importantly perhaps, the human and working capital built throughout the concertation process have the potential to be a fundamental cornerstone in dealing with more complex issues such as the chronically increasing level of underwater noise in whale habitats.

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.

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.

Subcutaneous immunization with recombinant adenovirus expressing influenza A nucleoprotein protects mice against lethal viral challenge.

Current influenza vaccines mainly induce strain-specific neutralizing antibodies and need to be updated each year, resulting in significant burdens on vaccine manufacturers and regulatory agencies. Genetic immunization strategies based on the highly conserved nucleoprotein (NP) of influenza have attracted great attention as NP could induce heterosubtypic immunity. It is unclear, however, whether different forms of vectors and/or vaccination regimens could have contributed to the previously reported discrepancies in the magnitude of protection of NP-based genetic vaccinations. Here, we evaluated a plasmid DNA vector (pNP) and a recombinant adenovirus vector (rAd-NP) containing the NP gene through various combinations of immunization regimens in mice. We found that pNP afforded only partial protection even after 4 injections, with full protection against lethal challenge achieved only with the fourth boost using rAd-NP. Alternatively, only two doses of rAd-NP delivered subcutaneously were needed to induce an enhanced immune response and completely protect the animals, a finding which, to our knowledge, has not been reported before.

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.