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1.  Superior In Vitro Stimulation of Human CD8+ T-Cells by Whole Virus versus Split Virus Influenza Vaccines 
PLoS ONE  2014;9(7):e103392.
Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8+ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8+ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4+ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8+ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8+ T cells should include whole virus rather than split virus formulations.
doi:10.1371/journal.pone.0103392
PMCID: PMC4114834  PMID: 25072749
2.  Preclinical evaluation of Vaxfectin®-adjuvanted Vero cell-derived seasonal split and pandemic whole virus influenza vaccines 
Human Vaccines & Immunotherapeutics  2013;9(6):1333-1345.
Increasing the potency and supply of seasonal and pandemic influenza vaccines remains an important unmet medical need which may be effectively accomplished with adjuvanted egg- or cell culture-derived vaccines. Vaxfectin®, a cationic lipid-based adjuvant with a favorable safety profile in phase 1 plasmid DNA vaccines trials, was tested in combination with seasonal split, trivalent and pandemic whole virus, monovalent influenza vaccines produced in Vero cell cultures. Comparison of hemagglutination inhibition (HI) antibody titers in Vaxfectin®-adjuvanted to nonadjuvanted vaccinated mice and guinea pigs revealed 3- to 20-fold increases in antibody titers against each of the trivalent influenza virus vaccine strains and 2- to 8-fold increases in antibody titers against the monovalent H5N1 influenza virus vaccine strain. With the vaccine doses tested, comparable antibody responses were induced with formulations that were freshly prepared or refrigerated at conventional 2–8°C storage conditions for up to 6 mo. Comparison of T-cell frequencies measured by interferon-gamma ELISPOT assay between groups revealed increases of between 2- to 10-fold for each of the adjuvanted trivalent strains and up to 22-fold higher with monovalent H5N1 strain. Both trivalent and monovalent vaccines were easy to formulate with Vaxfectin® by simple mixing. These preclinical data support further testing of Vaxfectin®-adjuvanted Vero cell culture vaccines toward clinical studies designed to assess safety and immunogenicity of these vaccines in humans.
doi:10.4161/hv.24209
PMCID: PMC3901827  PMID: 23857272
H5N1; Vaxfectin®; Vero cell culture; adjuvant; cationic liposomes; influenza vaccine
3.  Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model 
PLoS ONE  2012;7(9):e45842.
In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)3-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines.
doi:10.1371/journal.pone.0045842
PMCID: PMC3454321  PMID: 23029269
4.  A Cell Culture–Derived Influenza Vaccine Provides Consistent Protection Against Infection and Reduces the Duration and Severity of Disease in Infected Individuals 
A Vero cell culture–derived seasonal influenza vaccine provides consistently high levels of protection against cell culture–confirmed infection over a complete influenza season. Influenza symptoms are also less severe and of shorter duration in individuals who become infected despite vaccination.
Background. Current knowledge of the consistency of protection induced by seasonal influenza vaccines over the duration of a full influenza season is limited, and little is known about the clinical course of disease in individuals who become infected despite vaccination.
Methods. Data from a randomized double-blind placebo-controlled clinical trial undertaken in healthy young adults in the 2008–2009 influenza season were used to investigate the weekly cumulative efficacy of a Vero cell culture–derived influenza vaccine. In addition, the duration and severity of disease in vaccine and placebo recipients with cell culture–confirmed influenza infection were compared.
Results. Vaccine efficacy against matching strains was consistently high (73%–82%) throughout the study, including the entire period of the influenza season during which influenza activity was above the epidemic threshold. Vaccine efficacy was also consistent (68%–83%) when calculated for all strains, irrespective of antigenic match. Vaccination also ameliorated disease symptoms when infection was not prevented. Bivariate analysis of duration and severity showed a significant amelioration of myalgia (P = .003), headache (P = .025), and fatigue (P = .013) in infected vaccinated subjects compared with placebo. Cough (P = .143) and oropharyngeal pain (P = .083) were also reduced in infected vaccinated subjects.
Conclusions. A Vero cell culture–derived influenza vaccine provides consistently high levels of protection against cell culture–confirmed infection by seasonal influenza virus and significantly reduces the duration and severity of disease in those individuals in which infection is not prevented.
Clinical Trials Registration. ClinicalTrials.gov NCT00566345.
doi:10.1093/cid/cir959
PMCID: PMC3297649  PMID: 22267715
5.  H5N1 Whole-Virus Vaccine Induces Neutralizing Antibodies in Humans Which Are Protective in a Mouse Passive Transfer Model 
PLoS ONE  2011;6(8):e23791.
Background
Vero cell culture-derived whole-virus H5N1 vaccines have been extensively tested in clinical trials and consistently demonstrated to be safe and immunogenic; however, clinical efficacy is difficult to evaluate in the absence of wide-spread human disease. A lethal mouse model has been utilized which allows investigation of the protective efficacy of active vaccination or passive transfer of vaccine induced sera following lethal H5N1 challenge.
Methods
We used passive transfer of immune sera to investigate antibody-mediated protection elicited by a Vero cell-derived, non-adjuvanted inactivated whole-virus H5N1 vaccine. Mice were injected intravenously with H5N1 vaccine-induced rodent or human immune sera and subsequently challenged with a lethal dose of wild-type H5N1 virus.
Results
Passive transfer of H5N1 vaccine-induced mouse, guinea pig and human immune sera provided dose-dependent protection of recipient mice against lethal challenge with wild-type H5N1 virus. Protective dose fifty values for serum H5N1 neutralizing antibody titers were calculated to be ≤1∶11 for all immune sera, independently of source species.
Conclusions
These data underpin the confidence that the Vero cell culture-derived, whole-virus H5N1 vaccine will be effective in a pandemic situation and support the use of neutralizing serum antibody titers as a correlate of protection for H5N1 vaccines.
doi:10.1371/journal.pone.0023791
PMCID: PMC3158096  PMID: 21876771
6.  A Pandemic Influenza H1N1 Live Vaccine Based on Modified Vaccinia Ankara Is Highly Immunogenic and Protects Mice in Active and Passive Immunizations 
PLoS ONE  2010;5(8):e12217.
Background
The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus, and to show protection in a lethal animal challenge model.
Methodology/Principal Findings
For this purpose, the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus - a safe poxviral live vector – resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines, together with an inactivated whole virus vaccine, were assessed in a lung infection model using immune competent Balb/c mice, and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain, while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-γ-secreting (IFN-γ) CD4- and CD8 T-cells in lungs and spleens. In the lungs, a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus, which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition, passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus.
Conclusions/Significance
The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza.
doi:10.1371/journal.pone.0012217
PMCID: PMC2922371  PMID: 20808939
7.  A Whole Virus Pandemic Influenza H1N1 Vaccine Is Highly Immunogenic and Protective in Active Immunization and Passive Protection Mouse Models 
PLoS ONE  2010;5(2):e9349.
The recent emergence and rapid spread of a novel swine-derived H1N1 influenza virus has resulted in the first influenza pandemic of this century. Monovalent vaccines have undergone preclinical and clinical development prior to initiation of mass immunization campaigns. We have carried out a series of immunogenicity and protection studies following active immunization of mice, which indicate that a whole virus, nonadjuvanted vaccine is immunogenic at low doses and protects against live virus challenge. The immunogenicity in this model was comparable to that of a whole virus H5N1 vaccine, which had previously been demonstrated to induce high levels of seroprotection in clinical studies. The efficacy of the H1N1 pandemic vaccine in protecting against live virus challenge was also seen to be equivalent to that of the H5N1 vaccine. The protective efficacy of the H1N1 vaccine was also confirmed using a severe combined immunodeficient (SCID) mouse model. It was demonstrated that mouse and guinea pig immune sera elicited following active H1N1 vaccination resulted in 100% protection of SCID mice following passive transfer of immune sera and lethal challenge. The immune responses to a whole virus pandemic H1N1 and a split seasonal H1N1 vaccine were also compared in this study. It was demonstrated that the whole virus vaccine induced a balanced Th-1 and Th-2 response in mice, whereas the split vaccine induced mainly a Th-2 response and only minimal levels of Th-1 responses. These data supported the initiation of clinical studies with the same low doses of whole virus vaccine that had previously been demonstrated to be immunogenic in clinical studies with a whole virus H5N1 vaccine.
doi:10.1371/journal.pone.0009349
PMCID: PMC2826398  PMID: 20186321
8.  Nonreplicating Vaccinia Virus Vectors Expressing the H5 Influenza Virus Hemagglutinin Produced in Modified Vero Cells Induce Robust Protection▿  
Journal of Virology  2009;83(10):5192-5203.
The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system.
doi:10.1128/JVI.02081-08
PMCID: PMC2682067  PMID: 19279103
9.  Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses 
Vaccine  2007;25(32):6028-6036.
The rapid spread and the transmission to humans of avian influenza virus (H5N1) has induced world-wide fears of a new pandemic and raised concerns over the ability of standard influenza vaccine production methods to rapidly supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains were developed and demonstrated to be highly immunogenic in animal models. The vaccines induce cross-neutralising antibodies, highly cross-reactive T-cell responses and are protective in a mouse challenge model not only against the homologous virus but against other H5N1 strains, including those from another clade. These data indicate that cell culture-grown, whole virus vaccines, based on the wild-type virus, allow the rapid high yield production of a candidate pandemic vaccine.
doi:10.1016/j.vaccine.2007.05.013
PMCID: PMC2040225  PMID: 17614165
H5N1 whole virus vaccine; Vero cells

Results 1-9 (9)