Workshop report: Experimental animal models for universal influenza vaccines.

A major challenge in influenza research is the selection of an appropriate animal model that accurately reflects the disease and the protective immune response observed in humans. A workshop organised by the EDUFLUVAC consortium, a European Union funded project coordinated by the European Vaccine Initiative, brought together experts from the influenza vaccine community with the aim to discuss the current knowledge and future perspectives for testing broadly reactive influenza vaccines in animal models. The programme included a diversity of models from well-established and publicly accepted models to cutting edge, newly developed animal models as well as ex-vivo approaches and human models. The audience concluded that different vaccine approaches may require evaluation in different animal models, depending on the type of immune response induced by the vaccine. Safety is the main concern for transition to clinical development and influenza vaccine associated enhanced disease was specifically emphasised. An efficient animal model to evaluate this aspect of safety still needs to be identified. Working with animal models requires ethical compliance and consideration of the 3R principles. Development of alternative approaches such as ex-vivo techniques is progressing but is still at an early stage and these methods are not yet suitable for broader application for vaccine evaluation. The human challenge is the ultimate model to assess influenza vaccines. However this model is expensive and not largely applicable. The currently used pre-clinical models are not yet specifically focused on studying unique aspects of a universal influenza vaccine. Further collaboration, communication and effective networking are needed for success in establishment of harmonised and standardised pre-clinical models for evaluation of new influenza vaccines. This report does not provide a complete review of the field but discusses the data presented by the speakers and discussion points raised during the meeting.

Size distribution analysis of influenza virus particles using size exclusion chromatography.

Size exclusion chromatography is a standard method in quality control of biopharmaceutical proteins. In contrast, vaccine analysis is often based on activity assays. The hemagglutination assay is a widely accepted influenza quantification method, providing no insight in the size distribution of virus particles. Capabilities of size exclusion chromatography to complement the hemagglutination assay are investigated. The presented method is comparatively robust regarding different buffer systems, ionic strength and additive concentrations. Addition of 200mM arginine or sodium chloride is necessary to obtain complete virus particle recovery. 0.5 and 1.0M arginine increase the hydrodynamic radius of the whole virus particles by 5nm. Sodium citrate induces virus particle aggregation. Results are confirmed by dynamic light scattering. Retention of a H1N1v strain correlates with DNA contents between 5ng/mL and 670ng/mL. Quantitative elution of the virus preparations is verified on basis of hemagglutination activity. Elution of hemagglutination inducing compounds starts at a flow channel diameter of 7000nm. The universal applicability is demonstrated with three different influenza virus samples, including an industrially produced, pandemic vaccine strain. Size distribution of the pandemic H1N1v 5258, H1N1 PR/8/34, and H3N2 Aichi/2/68 preparations spreads across inter- and intra-particle volume and extends to the secondary interaction dominated range. Thus, virus particle debris seems to induce hemagglutination. Fragments generated by 0.5% Triton™ X-100 treatment increase overall hemagglutination activity.

Flucelvax (Optaflu) for seasonal influenza.

Conventional egg-based manufacturing technology for seasonal influenza vaccines has several drawbacks, including its inflexibility, reliance on egg supplies, risk of contamination, absence of growth of some isolates and egg-adaptive viral mutations that threaten vaccine matching. To overcome these limitations, cell culture-derived vaccines have been designed, including the trivalent inactivated vaccine Flucelvax®/Optaflu® (brand names in the US/EU, respectively). Flucelvax®/Optaflu® has gained wide regulatory approval and is currently implemented in several countries. Non-clinical studies have assuaged hypothetical concerns regarding oncogenicity and use in persons allergic to dogs. Ample clinical data suggest the non-inferiority of Flucelvax®/Optaflu® to egg-based vaccines in terms of immunogenicity, safety and tolerability, and it has fulfilled American and European mandatory requirements. Although Flucelvax®/Optaflu® is currently indicated only for adults and the elderly, pediatric data indicate its good immunogenicity and safety. This paper provides an update on the clinical development of Flucelvax®/Optaflu®, its seasonal trials and available post-marketing surveillance data.

Pandemic preparedness with live attenuated influenza vaccines based on A/Leningrad/134/17/57 master donor virus.

Continuously evolving avian influenza viruses pose a constant threat to the human public health. In response to this threat, a number of pandemic vaccine candidates have been prepared and evaluated in animal models and clinical trials. This review summarizes the data from the development and preclinical and clinical evaluation of pandemic live attenuated influenza vaccines (LAIV) based on Russian master donor virus A/Leningrad/134/17/57. LAIV candidates of H5N1, H5N2, H7N3, H1N1 and H2N2 subtypes were safe, immunogenic and protected animals from challenge with homologous and heterologous viruses. Clinical trials of the pandemic LAIVs demonstrated their safety and immunogenicity for healthy adult volunteers. The vaccine viruses were infectious, genetically stable and did not transmit to unvaccinated contacts. In addition, here we discuss criteria for the assessment of pandemic LAIV immunogenicity and efficacy necessary for their licensure.

Advances and challenges in the development and production of effective plant-based influenza vaccines.

Influenza infections continue to present a major threat to public health. Traditional modes of influenza vaccine manufacturing are failing to satisfy the global demand because of limited scalability and long production timelines. In contrast, subunit vaccines (SUVs) can be produced in heterologous expression systems in shorter times and at higher quantities. Plants are emerging as a promising platform for SUV production due to time efficiency, scalability, lack of harbored mammalian pathogens and possession of the machinery for eukaryotic post-translational protein modifications. So far, several organizations have utilized plant-based transient expression systems to produce SUVs against influenza, including vaccines based on virus-like particles. Plant-produced influenza SUV candidates have been extensively evaluated in animal models and some have shown safety and immunogenicity in clinical trials. Here, the authors review ongoing efforts and challenges to producing influenza SUV candidates in plants and discuss the likelihood of bringing these products to the market.

High-level systemic expression of conserved influenza epitope in plants on the surface of rod-shaped chimeric particles.

Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV) genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP) gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV) did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV­based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod­shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP) described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

The Madin-Darby canine kidney cell culture derived influenza A/H5N1 vaccine: a phase I trial in Taiwan.

BACKGROUND: Avian H5N1 influenza has caused human infections globally and has a high mortality rate. Rapid production of effective vaccines is needed. METHODS: A phase 1, randomized, observer-blinded clinical trial was conducted to examine the safety and immunogenicity of an inactivated whole virion vaccine against the influenza A/H5N1 virus produced from the Madin-Darby canine kidney (MDCK) cell line. Participants were randomized to four groups and administered two intramuscular doses of vaccine containing 3 µg hemagglutinin (HA), 3 µg HA with 300 µg aluminum phosphate (AlPO4), 6 µg HA, and 6 µg HA with 300 µg AlPO4, respectively, at two visits, 21 days apart. Serum hemagglutination inhibition (HAI) and neutralizing antibody levels were determined at baseline and on Days 21 and 42. RESULTS: Sixty healthy individuals were enrolled. The neutralization assay showed a significant immune response in the 6 µg with ALPO4 group on Day 42 compared to pre-vaccination levels (11.32±9.77 vs. 4.00±0, p=0.02). The adjuvant effect in neutralization assay was also significant on Day 42 in the 6 µg group (4.52±1.94 without adjuvant vs. 11.32±9.77 with adjuvant, p=0.02). HAI assay also showed an aluminum adjuvant-induced increasing trend in HAI geometric mean titer on Day 42 in the 3 µg and 6 µg groups (6.02 versus 8.20, p=0.05 and 5.74 versus 8.21, p=0.14). The most frequent adverse event was local pain (20% to 60%). There were no vaccine-related severe adverse effects. CONCLUSION: MDCK cell line-derived H5N1 vaccine was well tolerated. It is necessary to investigate further the immunogenicity of higher antigen doses and the role of aluminum adjuvant in augmenting the effect of the vaccine.

Sulfated membrane adsorbers for economic pseudo-affinity capture of influenza virus particles.

Strategies to control outbreaks of influenza, a contagious respiratory tract disease, are focused mainly on prophylactic vaccinations in conjunction with antiviral medications. Currently, several mammalian cell culture-based influenza vaccine production processes are being established, such as the technologies introduced by Novartis Behring (Optaflu) or Baxter International Inc. (Celvapan). Downstream processing of influenza virus vaccines from cell culture supernatant can be performed by adsorbing virions onto sulfated column chromatography beads, such as Cellufine sulfate. This study focused on the development of a sulfated cellulose membrane (SCM) chromatography unit operation to capture cell culture-derived influenza viruses. The advantages of the novel method were demonstrated for the Madin Darby canine kidney (MDCK) cell-derived influenza virus A/Puerto Rico/8/34 (H1N1). Furthermore, the SCM-adsorbers were compared directly to column-based Cellufine sulfate and commercially available cation-exchange membrane adsorbers. Sulfated cellulose membrane adsorbers showed high viral product recoveries. In addition, the SCM-capture step resulted in a higher reduction of dsDNA compared to the tested cation-exchange membrane adsorbers. The productivity of the SCM-based unit operation could be significantly improved by a 30-fold increase in volumetric flow rate during adsorption compared to the bead-based capture method. The higher flow rate even further reduced the level of contaminating dsDNA by about twofold. The reproducibility and general applicability of the developed unit operation were demonstrated for two further MDCK cell-derived influenza virus strains: A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004. Overall, SCM-adsorbers represent a powerful and economically favorable alternative for influenza virus capture over conventional methods using Cellufine sulfate.

Pinellic acid from the tuber of Pinellia ternata Breitenbach as an effective oral adjuvant for nasal influenza vaccine.

This study describes the isolation, purification, characterization, and adjuvant activity of an orally active adjuvant substance from the tuber of Pinellia ternata, as an active herbal component of the traditional Japanese herbal (Kampo) medicine, Sho-seiryu-to (SST, Chinese name: Xiao-Qing-Long-Tang), which has been reported to show oral adjuvant activity for nasally administered influenza HA vaccine [Int. J. Immunopharmacol. 16 (1994) 605]. The active compound was identified as 9S, 12S, 13S-trihydroxy-10E-octadecenoic acid using infrared spectra, proton magnetic resonance, mass spectrometry, and circular dichroism, and named pinellic acid. Oral administration of pinellic acid (1 microg) to BALB/c mice given primary and secondary intranasal inoculations of influenza HA vaccine (1 microg) enhanced antiviral IgA antibody (Ab) titers 5.2- and 2.5-fold in nasal and bronchoalveolar washes, respectively, and antiviral IgG Ab titers 3-fold in bronchoalveolar wash and serum. Intranasal administration of pinellic acid (1 microg) with influenza HA vaccine (1 microg) slightly enhanced antiviral IgG Ab titers in bronchoalveolar wash and serum but not antiviral IgA Ab titers in nasal and bronchoalveolar washes. Pinellic acid showed no hemolytic activity. The results of this study suggest that pinellic acid may provide a safe and potent oral adjuvant for nasal influenza HA vaccine.

The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines.

Influenza viruses for vaccine production are currently grown on embryonated eggs. This manufacturing system conveys many major drawbacks such as inflexibility, cumbersome down stream processing, inability of some strains to replicate on eggs to high enough yields, and selection of receptor-binding variants with reduced antigenicity. These limitations emphasize the need for a cell line-based production system that could replace eggs in the production of influenza virus vaccines in a pandemic proof fashion. Here we present the efficient propagation of influenza A and B viruses on the fully characterized and standardized human cell line PER.C6.

[The potentials for immunization against influenza using liposome-incorporated viral surface antigens]. / Possibilités d'immunisation contre la grippe à l'aide d'antigènes viraux de surface incorporés aux liposomes.

Studies were conducted using uni- and multilamellar liposomes to establish optimum conditions for influenza antigen incorporation in view of their transport to the target cells for experimental influenza prophylaxis in hybrid white mice. Radiometric determinations showed a good level of preparation purification, a good efficiency of incorporation in liposomes of the active biological material, the liposome linked radioactivity distribution among different organs. Charged liposomes induced solid and long lasting resistance against influenza control infection.

[Immunogenicity of a subunit influenza vaccine in experiments on animals]. / Izuchenie immunogennosti sub"edinichnoi grippoznoi vaktsiny v opytakh na zhivotnykh.

The data on isolation of purified influenza virus glycoproteins and their reconstruction into liposomes by means of a new nonionic detergent, MESK, are presented. Sedimentational and flotational distribution of glycoprotein and liposome preparations was studied. In the presence of the detergent, the isolated glycoproteins were shown to occur mostly in a monomeric form. Removal of the detergent by dialysis resulted in formation of protein micelles, and the presence of exogenously introduced lipids in reconstruction of liposomes heterogeneous in composition. The resulting preparations had a high degree of biochemical purity and biological activity meeting the requirements for subunit vaccine properties. The immunogenic potency of a subunit influenza vaccine produced with the use of the MESK detergent was studied. The glycoproteins isolated with the use of MESK were shown to be comparable in their immunogenic potency with glycoproteins comprising viral particles. The influence of the form of glycoproteins presentation on their immunogenic potency was studied. Glycoproteins in the form of micelles and as components of liposomes were found to have a good immunogenic potency and to be able to induce protective immunity preventing experimental influenza infection in mice. Monomeric forms of glycoproteins had no such properties.

[Immunization with a complex preparation: a new method of influenza prevention. The basis for using a complex preparation for preventing influenza]. / Immunizatsiia kompleksnym preparatom--novyi metod profilaktiki grippa. Obosnovanie primeneniia kompeksnogo preparata dlia profilaktiki grippoznoi infektsii.

A combined preparation for influenza prevention (CPIP) consisting of an interferon inducer stimulating immunogenesis and killed influenza vaccine is proposed. Twenty five inducers-stimulators have been tested: polynucleotides, polysaccharide and lipopolysaccharide extracted from Salmonella typhosa. Intranasal administration of CPIP to laboratory animals markedly stimulates interferon, secretory, and circulating antibody synthesis. Resistance to fatal influenza infection develops within 18 hours after administration of CPIP and its intensity increases in the following 14 days (the observation period.

Alkaline-extracted influenza subunit vaccine.

Treatment of influenza virus concentrates with alkaline solvents releases a major fraction of the viral structural protein content. As determined by polyacrylamide gel electrophoresis, the surface glycoprotein substructures, hemagglutinin and neuraminidase, are the primary solubilized products. Two forms of hemagglutinin antigen are recovered, a 39S active hemagglutinin and a 23S blocking antigen. Dose-response assays in mice demonstrate that hemagglutination-inhibiting and neuraminidase antibodies are induced. Antibody responses are comparable to those resulting from immunization with inactivated whole virus. On the basis of demonstrated purity, high yields of protective antigens, immunogenic potency, and absence of deleterious reagents, alkaline-extracted influenza protein preparations merit consideration as subunit vaccines for human use.

[Characteristics of the basic parameters of killed influenza vaccine purified and concentrated on sodium borsilicate glass]. / Kharakteristike osnovnykh parametrov ubitoi grippoznoi vaktsiny, ochishchennoi i kontsentrirovannoi na natriiborsilikatnom stekle

The main parameters (reactogenicity, harmlessness, immunogenicity and prophylactic efficacy) of experimental batches of the inactivated influenza vaccine A, purified and concentrated on sodium-boron-silicate glass, were studied in strictly controlled epidemiological trial. In single subcutaneous injection in a dose of 0.5 ml (5120 hemagglutinating units) of the vaccine it was characterized by a moderate reactogenicity, marked antigenicity and prophylactic, efficacy.