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1.  Phase I/II Randomized Double-Blind Study of the Safety and Immunogenicity of a Nonadjuvanted Vero Cell Culture-Derived Whole-Virus H9N2 Influenza Vaccine in Healthy Adults 
Studies on candidate pandemic vaccines against avian influenza viruses have focused on H5N1, but viruses of other subtypes, such as A/H9N2, are also considered to have pandemic potential. We investigated the safety and immunogenicity of two immunizations with one of five different antigen doses (ranging from 3.75 to 45 μg of hemagglutinin antigen) of a nonadjuvanted whole-virus G9 lineage H9N2 influenza virus vaccine in healthy adults aged 18 to 49 years. The antibody responses were measured by hemagglutination inhibition (HI), microneutralization (MN), and single radial hemolysis (SRH) assays. To investigate a hypothesis that previous exposure to H2N2 viruses in subjects born in or before 1968 might prime for more robust antibody responses to H9N2 vaccination than that in subjects born after 1968, a post hoc age-stratified analysis of antibody responses was done. Both vaccinations in all dose groups were safe and well tolerated. No vaccine-related serious adverse events were reported, and the majority of the adverse reactions were rated as mild. The rates of injection site reactions were lower in the 3.75-μg- and 7.5-μg-dose groups than those in the higher-dose groups; the rates of systemic reactions were similar across all dose groups. The seroprotection rates among the different dose groups 21 days after the second immunization ranged from 52.8% to 88.9% as measured by HI assay, from 88.7% to 98.1% or 82.7% to 96.2% as measured by MN assay (MN titer cutoffs, 1:40 and 1:80, respectively), and from 94.2% to 100% as measured by SRH assay. Higher antibody responses were not induced in subjects born in or before 1968. These data indicate that a nonadjuvanted whole-virus H9N2 vaccine is well tolerated and immunogenic in healthy adults. (This study has been registered at under registration no. NCT01320696.)
PMCID: PMC4278922  PMID: 25355797
2.  A Novel Multivalent OspA Vaccine against Lyme Borreliosis Is Safe and Immunogenic in an Adult Population Previously Infected with Borrelia burgdorferi Sensu Lato 
Clinical and Vaccine Immunology : CVI  2014;21(11):1490-1499.
Lyme borreliosis (LB) patients who recover, as well as previously infected asymptomatic individuals, remain vulnerable to reinfection with Borrelia burgdorferi sensu lato. There is limited information available about the use of OspA vaccines in this population. In this study, a randomized double-blind phase I/II trial was performed to investigate the safety and immunogenicity of a novel multivalent OspA vaccine in healthy adults who were either seronegative or seropositive for previous B. burgdorferi sensu lato infection. The participants received three monthly priming immunizations with either 30 μg or 60 μg alum-adjuvanted OspA antigen and a booster vaccination either 6 months or 9 to 12 months after the first immunization. The antibody responses to the six OspA serotypes included in the vaccine were evaluated. Adverse events were predominantly mild and transient and were similar in the seronegative and seropositive populations. Substantial enzyme-linked immunosorbent assay (ELISA) and surface-binding antibody responses against all six OspA antigens were induced after the primary immunization schedule in both populations, and they were substantially increased with both booster schedules. The antibody responses induced by the two doses were similar in the seronegative population, but there was a significant dose response in the seropositive population. These data indicate that the novel multivalent OspA vaccine is well tolerated and immunogenic in individuals previously infected with B. burgdorferi sensu lato. (This study is registered at under registration no. NCT01504347.)
PMCID: PMC4248771  PMID: 25185574
3.  Immunogenicity and Protective Efficacy of a Vero Cell Culture-Derived Whole-Virus H7N9 Vaccine in Mice and Guinea Pigs 
PLoS ONE  2015;10(2):e0113963.
A novel avian H7N9 virus with a high case fatality rate in humans emerged in China in 2013. We evaluated the immunogenicity and protective efficacy of a candidate Vero cell culture-derived whole-virus H7N9 vaccine in small animal models.
Antibody responses induced in immunized DBA/2J mice and guinea pigs were evaluated by hemagglutination inhibition (HI), microneutralization (MN), and neuraminidase inhibition (NAi) assays. T-helper cell responses and IgG subclass responses in mice were analyzed by ELISPOT and ELISA, respectively. Vaccine efficacy against lethal challenge with wild-type H7N9 virus was evaluated in immunized mice. H7N9-specific antibody responses induced in mice and guinea pigs were compared to those induced by a licensed whole-virus pandemic H1N1 (H1N1pdm09) vaccine.
The whole-virus H7N9 vaccine induced dose-dependent H7N9-specific HI, MN and NAi antibodies in mice and guinea pigs. Evaluation of T-helper cell responses and IgG subclasses indicated the induction of a balanced Th1/Th2 response. Immunized mice were protected against lethal H7N9 challenge in a dose-dependent manner. H7N9 and H1N1pdm09 vaccines were similarly immunogenic.
The induction of H7N9-specific antibody and T cell responses and protection against lethal challenge suggest that the Vero cell culture-derived whole-virus vaccine would provide an effective intervention against the H7N9 virus.
PMCID: PMC4342221  PMID: 25719901
4.  Safety and Immunogenicity of a Vero Cell Culture-Derived Whole-Virus H5N1 Influenza Vaccine in Chronically Ill and Immunocompromised Patients 
The development of vaccines against H5N1 influenza A viruses is a cornerstone of pandemic preparedness. Clinical trials of H5N1 vaccines have been undertaken in healthy subjects, but studies in risk groups have been lacking. In this study, the immunogenicity and safety of a nonadjuvanted cell culture-derived whole-virus H5N1 vaccine were assessed in chronically ill and immunocompromised adults. Subjects received two priming immunizations with a clade 1 A/Vietnam H5N1 influenza vaccine, and a subset also received a booster immunization with a clade 2.1 A/Indonesia H5N1 vaccine 12 to 24 months later. The antibody responses in the two populations were assessed by virus neutralization and single radial hemolysis assays. The T-cell responses in a subset of immunocompromised patients were assessed by enzyme-linked immunosorbent spot assay (ELISPOT). The priming and the booster vaccinations were safe and well tolerated in the two risk populations, and adverse reactions were predominantly mild and transient. The priming immunizations induced neutralizing antibody titers of ≥1:20 against the A/Vietnam strain in 64.2% of the chronically ill and 41.5% of the immunocompromised subjects. After the booster vaccination, neutralizing antibody titers of ≥1:20 against the A/Vietnam and A/Indonesia strains were achieved in 77.5% and 70.8%, respectively, of chronically ill subjects and in 71.6% and 67.5%, respectively, of immunocompromised subjects. The T-cell responses against the two H5N1 strains increased significantly over the baseline values. Substantial heterosubtypic T-cell responses were elicited against the 2009 pandemic H1N1 virus and seasonal A(H1N1), A(H3N2), and B subtypes. There was a significant correlation between T-cell responses and neutralizing antibody titers. These data indicate that nonadjuvanted whole-virus cell culture-derived H5N1 influenza vaccines are suitable for immunizing chronically ill and immunocompromised populations. (This study is registered at under registration no. NCT00711295.)
PMCID: PMC4054238  PMID: 24739978
5.  Neuraminidase-Inhibiting Antibody Response to H5N1 Virus Vaccination in Chronically Ill and Immunocompromised Patients 
Open Forum Infectious Diseases  2014;1(2):ofu072.
Neuraminidase-inhibiting (NAi) antibodies have been reported to be an independent correlate of protection from influenza disease, but the NAi antibody response to influenza vaccination has never been assessed in chronically ill or immunocompromised participants. Using an enzyme-linked lectin assay, we demonstrated that 2 immunizations with a Vero cell culture-derived, whole-virus H5N1 A/Vietnam vaccine induces NAi antibodies in 94.3% of chronically ill and 83.8% of immunocompromised participants. A booster with a heterologous A/Indonesia H5N1 vaccine induced comparable NAi antibody titers in both groups and resulted in 100% seropositivity. These data support prepandemic H5N1 vaccination strategies for these highly vulnerable risk groups.
PMCID: PMC4281780  PMID: 25734142
avian influenza; vero cell culture; influenza vaccine; neuraminidase; risk populations
6.  Hyperimmune intravenous immunoglobulin containing high titers of pandemic H1N1 hemagglutinin and neuraminidase antibodies provides dose-dependent protection against lethal virus challenge in SCID mice 
Virology Journal  2014;11:70.
Convalescent plasma and fractionated immunoglobulins have been suggested as prophylactic or therapeutic interventions during an influenza pandemic.
Intravenous immunoglobulin (IVIG) preparations manufactured from human plasma collected before the 2009 H1N1 influenza pandemic, and post-pandemic hyperimmune (H)-IVIG preparations were characterized with respect to hemagglutination inhibition (HI), microneutralization (MN) and neuraminidase-inhibiting (NAi) antibody titers against pandemic H1N1 (pH1N1) and seasonal H1N1 (sH1N1) viruses. The protective efficacy of the IVIG and H-IVIG preparations was evaluated in a SCID mouse challenge model.
Substantial levels of HI, MN and NAi antibodies against pH1N1 (GMTs 1:45, 1:204 and 1: 727, respectively) and sH1N1 (GMTs 1:688, 1:4,946 and 1:312, respectively) were present in pre-pandemic IVIG preparations. In post-pandemic H-IVIG preparations, HI, MN and NAi antibody GMTs against pH1N1 were 1:1,280, 1:11,404 and 1:2,488 (28-, 56- and 3.4-fold enriched), respectively, compared to pre-pandemic IVIG preparations (p < 0.001). Post-pandemic H-IVIG (HI titer 1:1,280) provided complete protection from lethality of SCID mice against pH1N1 challenge (100% of mice survived for 29 days post-challenge). Pre-pandemic IVIG (HI titer 1:70) did not provide significant protection against pH1N1 challenge (50% of mice survived 29 days post-challenge compared to 40% survival in the buffer control group). There was a highly significant correlation between circulating in vivo HI and MN antibody titers and survival (p < 0001).
The substantial enrichment of HA- and NA-specific antibodies in H-IVIG and the efficacious protection of SCID mice against challenge with pH1N1 suggests H-IVIG as a promising intervention against pandemic influenza for immunocompromised patients and other risk groups.
PMCID: PMC3996311  PMID: 24739285
H1N1; IVIG; Influenza; Intravenous immunoglobulin; Passive transfer; Neutralizing antibody; Neuraminidase; Hemagglutinin
7.  MVA Vectors Expressing Conserved Influenza Proteins Protect Mice against Lethal Challenge with H5N1, H9N2 and H7N1 Viruses 
PLoS ONE  2014;9(2):e88340.
The availability of a universal influenza vaccine able to induce broad cross-reactive immune responses against diverse influenza viruses would provide an alternative to currently available strain-specific vaccines. We evaluated the ability of vectors based on modified vaccinia virus Ankara (MVA) expressing conserved influenza proteins to protect mice against lethal challenge with multiple influenza subtypes.
Mice were immunized with MVA vectors expressing H5N1-derived nucleoprotein (NP), the stem region of hemagglutinin (HA), matrix proteins 1 and 2 (M1 and M2), the viral polymerase basic protein 1 (PB1), or the HA stem fused to a quadrivalent matrix protein 2 extracellular domain (M2e). Immunized mice were challenged with lethal doses of H5N1, H7N1 or H9N2 virus and monitored for disease symptoms and weight loss. To investigate the influence of previous exposure to influenza virus on protective immune responses induced by conserved influenza proteins, mice were infected with pandemic H1N1 virus (H1N1pdm09) prior to immunization and subsequently challenged with H5N1 virus. Antibody and T cell responses were assessed by ELISA and flow cytometry, respectively.
MVA vectors expressing NP alone, or co-expressed with other conserved influenza proteins, protected mice against lethal challenge with H5N1, H7N1 or H9N2 virus. Pre-exposure to H1N1pdm09 increased protective efficacy against lethal H5N1 challenge. None of the other conserved influenza proteins provided significant levels of protection against lethal challenge. NP-expressing vectors induced high numbers of influenza-specific CD4+ and CD8+ T cells and high titer influenza-specific antibody responses. Higher influenza-specific CD4+ T cell responses and NP-specific CD8+ T cell responses were associated with increased protective efficacy.
MVA vectors expressing influenza NP protect mice against lethal challenge with H5N1, H7N1 and H9N2 viruses by a mechanism involving influenza-specific CD4+ and CD8+ T cell responses.
PMCID: PMC3921149  PMID: 24523886
8.  Evaluation of OspA Vaccination-Induced Serological Correlates of Protection against Lyme Borreliosis in a Mouse Model 
PLoS ONE  2013;8(11):e79022.
For clinical development of a novel multivalent OspA vaccine against Lyme borreliosis, serological assays are required which can be used to establish immune correlates of protection against infection with Borrelia.
Four assays (an OspA IgG ELISA, a competitive inhibition (CI) ELISA, a Borrelia surface-binding (SB) assay and a Borrelia killing assay) were used to evaluate the correlation between immune responses induced by rOspA 1/2 (a chimeric immunogen containing protective epitopes from OspA serotypes 1 and 2), and protective immunity against infection by B. burgdorferi s.s. (OspA-1) and B. afzelii (OspA-2). Mice were immunized with OspA 1/2 doses ranging from 0.3 ng to 100 ng, to induce a range of OspA antibody titers, and exposed to needle challenge with B. burgdorferi s.s. or tick challenge with B. afzelii. Receiver operator characteristics (ROC) curves were constructed for each assay, and the area under the curve (AUC), sensitivity, specificity and Youden Index were calculated. Potential cutoff antibody titers which could be used as correlates of vaccine-induced protection were derived from the maximum Youden Index.
Immunization with OspA-1/2 provided dose-dependent protection against infection with B. burgdorferi s.s. and B. afzelii. Antibody responses detected by all four assays were highly significantly correlated with protection from infection by either B. burgdorferi s.s. (p<0.0001 to 0.0062) or B. afzelii (p<0.0001). ROC analyses of the diagnostic effectiveness of each assay showed the AUC to range between 0.95 and 0.79, demonstrating that all assays distinguish well between infected and non-infected animals. Based on sensitivity, specificity and AUC, the OspA IgG ELISA and SB assays best discriminated between infected and non-infected animals.
All four assays differentiate well between Borrelia-infected and non-infected animals. The relatively simple, high throughput IgG ELISA would be suitable to establish immune correlates of protection for the novel OspA vaccine in clinical trials.
PMCID: PMC3832494  PMID: 24260146
9.  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. NCT00566345.
PMCID: PMC3297649  PMID: 22267715
10.  H5N1 Whole-Virus Vaccine Induces Neutralizing Antibodies in Humans Which Are Protective in a Mouse Passive Transfer Model 
PLoS ONE  2011;6(8):e23791.
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.
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.
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.
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.
PMCID: PMC3158096  PMID: 21876771
11.  Influence of vector design and host cell on the mechanism of recombination and emergence of mutant subpopulations of replicating retroviral vectors 
The recent advent of murine leukaemia virus (MLV)-based replication-competent retroviral (RCR) vector technology has provided exciting new tools for gene delivery, albeit the advances in vector efficiency which have been realized are also accompanied by a set of fresh challenges. The expression of additional transgene sequences, for example, increases the length of the viral genome, which can lead to reductions in replication efficiency and in turn to vector genome instability. This necessitates efforts to analyse the rate and mechanism of recombinant emergence during the replication of such vectors to provide data which should contribute to improvements in RCR vector design.
In this study, we have performed detailed molecular analyses on packaged vector genomes and proviral DNA following propagation of MLV-based RCR vectors both in cell culture and in pre-formed subcutaneous tumours in vivo. The effects of strain of MLV, transgene position and host cell type on the rate of emergence of vector recombinants were quantitatively analysed by applying real-time PCR and real-time RT-PCR assays. Individual mutants were further characterized by PCR, and nucleotide sequence and structural motifs associated with these mutants were determined by sequencing. Our data indicate that virus strain, vector design and host cell influence the rate of emergence of predominating vector mutants, but not the underlying recombination mechanisms in vitro. In contrast, however, differences in the RNA secondary structural motifs associated with sequenced mutants emerging in cell culture and in solid tumours in vivo were observed.
Our data provide further evidence that MLV-based RCR vectors based on the Moloney strain of MLV and containing the transgene cassette in the 3' UTR region are superior to those based on Akv-MLV and/or containing the transgene cassette in the U3 region of the LTR. The observed discrepancies between the data obtained in solid tumours in vivo and our own and previously published data from infected cells in vitro demonstrates the importance of evaluating vectors designed for use in cancer gene therapy in vivo as well as in vitro.
PMCID: PMC2645402  PMID: 19203366
12.  Transgene Expression Facilitated by the v-src Splice Acceptor Can Impair Replication Kinetics and Lead to Genomic Instability of Rous Sarcoma Virus-Based Vectors▿ † 
Journal of Virology  2007;82(3):1610-1614.
Rous sarcoma virus (RSV) can be used for the simple generation of high-titer replication-competent retroviral (RCR) vectors. Retroviruses undergo frequent genomic recombination, however, and vectors with reduced replication kinetics are rapidly overgrown by mutant forms. Vector design is hence critical to vector efficacy. In this study, two different designs of RSV-based RCR vectors were evaluated. Vectors in which transgene expression was facilitated by the v-src splice acceptor were revealed to have greatly reduced replication kinetics and genomic stability in comparison to vectors in which transgene expression was mediated by an internal ribosome entry site in the 3′ untranslated region.
PMCID: PMC2224446  PMID: 18057258
13.  Effects of Viral Strain, Transgene Position, and Target Cell Type on Replication Kinetics, Genomic Stability, and Transgene Expression of Replication-Competent Murine Leukemia Virus-Based Vectors▿  
Journal of Virology  2007;81(13):6973-6983.
The limited efficiency of in vivo gene transfer by replication-deficient retroviral vectors remains an obstacle to achieving effective gene therapy for solid tumors. One approach to circumvent this problem is the use of replication-competent retroviral vectors. However, the application of such vectors is at a comparatively early stage and the effects which virus strain, transgene cassette position, and target cell can exert on vector spread kinetics, genomic stability, and transgene expression levels remain to be fully elucidated. Thus, in this study a panel of vectors allowing the investigation of different design features on an otherwise genetically identical background were analyzed with respect to these readout parameters in cultures of both murine and human cells and in preformed tumors in nude mice. The obtained data revealed that (i) Moloney murine leukemia virus (Mo-MLV)-based vectors spread with faster kinetics, drive higher levels of transgene expression, and are more stable than equivalent Akv-MLV-based vectors; (ii) vectors containing the transgene cassette directly downstream of the envelope gene are genomically more stable than those containing it within the 3′-long terminal repeat U3 region; and (iii) the genomic stability of both strains seems to be cell line dependent.
PMCID: PMC1933297  PMID: 17442710
14.  Tissue- and Tumor-Specific Targeting of Murine Leukemia Virus-Based Replication-Competent Retroviral Vectors 
Journal of Virology  2006;80(14):7070-7078.
Replication-competent retrovirus vectors based on murine leukemia virus (MLV) have been shown to effectively transfer therapeutic genes over multiple serial infections in cell culture and through solid tumors in vivo with a high degree of genomic stability. While simple retroviruses possess a natural tumor selectivity in that they can transduce only actively dividing cells, additional tumor-targeting strategies would nevertheless be advantageous, since tumor cells are not the only actively dividing cells. In this study, we used the promiscuous murine cytomegalovirus promoter, a chimeric regulatory sequence consisting of the hepatitis B virus enhancer II and the human α1-antitrypsin (EII-Pa1AT) promoter, and a synthetic regulatory sequence consisting of a series of T-cell factor binding sites named the CTP4 promoter to generate replicating MLV vectors, whereby the last two are transcriptionally restricted to liver- and β-catenin/T-cell factor-deregulated cells, respectively. When the heterologous promoters were used to replace almost the entire MLV U3 region, including the MLV TATA box, vector replication was inefficient since nascent virus particle production from infected cells was greatly decreased. Fusion of the heterologous promoters lacking the TATA box to the MLV TATA box, however, generated vectors which replicated with almost-wild-type kinetics throughout permissive cells while exhibiting low or negligible spread in nonpermissive cells. The genomic stability of the vectors was shown to be comparable to that of a similar vector containing wild-type MLV long terminal repeats, and tropism analysis over repeated infection cycles showed that the targeted vectors retained their original specificity.
PMCID: PMC1489065  PMID: 16809312

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