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.

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.

Background

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.

Results

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.

Conclusion

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.

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.

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.

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.