Elevated Epstein-Barr virus loads and lower antibody titers in competitive athletes.

Epstein-Barr virus (EBV) is a persisting herpesvirus which is controlled by the adaptive immune response after primary infection and maintained in a latent state. However, reactivation or persistent replication is observed in situations where the immune response is compromised. Since intensive physical training has been reported to diminish immune function, increased EBV load may be a cause of reduced performance and decreased ability to sustain high training loads in competitive athletes. Samples drawn from 209 athletes during their regular follow-up appointments were tested. One hundred sixty-five individuals of similar age not active in competitive sports served as case-controls. EBV load was quantified in peripheral blood leucocytes (PBLs) by real-time PCR, and EBV antibodies were detected in plasma by ELISA and immunoblot analysis. EBV DNA was detectable in 25 of 209 athletes and in 26 of 165 controls. Of note, the EBV load per 10(5) PBLs was 6.44 +/- 1.75 in the case and 1.67 +/- 0.44 copies in the controls, yielding a high significant difference (P < 0.0001). However, EBV-specific IgG titers were significantly lower in athletes (150.4 +/- 10.73 U ml(-1) vs. 241.6 +/- 18.59 U ml(-1)). As monitored by immunoblotting, primary infections were detected with low prevalence, three in the case group and one in the control group. These findings demonstrate that EBV is present at higher levels in athletes, but the antibody response is lower in athletes than in the controls. J. Med. Virol. 82:446-451, 2010. (c) 2010 Wiley-Liss, Inc.

Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma are associated with nonprogressive HIV-1 infection.

BACKGROUND: Long-term survival of HIV-1 infected individuals is usually achieved by continuous administration of combination antiretroviral therapy (ART). An exception to this scenario is represented by HIV-1 infected nonprogressors (NP) which maintain relatively high circulating CD4+ T cells without clinical symptoms for several years in the absence of ART. Several lines of evidence indicate an important role of the T-cell response in the modulation of HIV-1 infection during the acute and chronic phase of the disease. RESULTS: We analyzed the functional and the differentiation phenotype of Nef- and Tat-specific CD8+ T cells in a cohort of HIV-1 infected NP in comparison to progressors, ART-treated seropositive individuals and individuals undergoing a single cycle of ART interruption. We observed that a distinctive feature of NP is the presence of Nef-specific CD45RA+ CD8+ T cells secreting MIP-1beta but not IFN-gamma. This population was present in 7 out of 11 NP. CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells were not detected in HIV-1 infected individuals under ART or withdrawing from ART and experiencing a rebounding viral replication. In addition, we detected Nef-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cells in only 1 out of 10 HIV-1 infected individuals with untreated progressive disease. CONCLUSION: The novel antigen-specific CD45RA+ IFN-gammaneg MIP-1beta+ CD8+ T cell population represents a new candidate marker of long-term natural control of HIV-1 disease progression and a relevant functional T-cell subset in the evaluation of the immune responses induced by candidate HIV-1 vaccines.

Therapeutic vaccination reduces HIV sequence variability.

With HIV persisting lifelong in infected persons, therapeutic vaccination is a novel alternative concept to control virus replication. Even though CD8 and CD4 cell responses to such immunizations have been demonstrated, their effects on virus replication are still unclear. In view of this fact, we studied the impact of a therapeutic vaccination with HIV nef delivered by a recombinant modified vaccinia Ankara vector on viral diversity. We investigated HIV sequences derived from chronically infected persons before and after therapeutic vaccination. Before immunization the mean +/- se pairwise variability of patient-derived Nef protein sequences was 0.1527 +/- 0.0041. After vaccination the respective value was 0.1249 +/- 0.0042, resulting in a significant (P<0.0001) difference between the two time points. The genes vif and 5'gag tested in parallel and nef sequences in control persons yielded a constant amino acid sequence variation. The data presented suggest that Nef immunization induced a selective pressure, limiting HIV sequence variability. To our knowledge this is the first report directly linking therapeutic HIV vaccination to decreasing diversity in patient-derived virus isolates.

Marker gene swapping facilitates recombinant Modified Vaccinia Virus Ankara production by host-range selection.

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental and human vaccine vector for its lack of replication in mammalian cells and high expression of heterologous genes. Recombinant MVA technology can be improved greatly by combining transient host-range selection (based on the restoration in MVA of the deleted vaccinia gene K1L) with the differential expression of fluorescent proteins. Recombinant virus results from swapping a red protein gene (in the acceptor virus) with a cassette of the transfer plasmid comprising the transgene and the green marker K1Lgfp (a chimeric gene comprising K1L and EGFP). Recombinant selection is performed in the selective host RK13. Finally, in the non-selective host BHK-21, a single crossover between identical flanking regions excises the marker gene. The three types of viruses involved (red parental, green intermediate and colourless final recombinant) are visualized differentially by fluorescence microscopy or fluoro-imaging of terminal dilution microcultures, leading to a straightforward and efficient purification protocol. This method (Red-to-Green gene swapping) reduces greatly the time needed to obtain marker-free recombinant MVA and increases the reliability of the construction process.

Nef-induced CCL2 Expression Contributes to HIV/SIV Brain Invasion and Neuronal Dysfunction.

C-C motif chemokine ligand 2 (CCL2) is a chemoattractant for leukocytes including monocytes, T cells, and natural killer cells and it plays an important role in maintaining the integrity and function of the brain. However, there is accumulating evidence that many neurological diseases are attributable to a dysregulation of CCL2 expression. Acquired immune deficiency syndrome (AIDS) encephalopathy is a severe and frequent complication in individuals infected with the human immunodeficiency virus (HIV) or the simian immunodeficiency virus (SIV). The HIV and SIV Nef protein, a progression factor in AIDS pathology, can be transferred by microvesicles including exosomes and tunneling nanotubes (TNT) within the host even to uninfected cells, and Nef can induce CCL2 expression. This review focuses on findings which collectively add new insights on how Nef-induced CCL2 expression contributes to neurotropism and neurovirulence of HIV and SIV and elucidates why adjuvant targeting of CCL2 could be a therapeutic option for HIV-infected persons.

The intracellular detection of MIP-1beta enhances the capacity to detect IFN-gamma mediated HIV-1-specific CD8 T-cell responses in a flow cytometric setting providing a sensitive alternative to the ELISPOT.

BACKGROUND: T-cell mediated immunity likely plays an important role in controlling HIV-1 infection and progression to AIDS. Several candidate vaccines against HIV-1 aim at stimulating cellular immune responses, either alone or together with the induction of neutralizing antibodies, and assays able to measure CD8 and CD4 T-cell responses need to be implemented. At present, the IFN-gamma-based ELISPOT assay is considered the gold standard and it is broadly preferred as primary assay for detection of antigen-specific T-cell responses in vaccine trials. However, in spite of its high sensitivity, the measurement of the sole IFN-gamma production provides limited information on the quality of the immune response. On the other hand, the introduction of polychromatic flow-cytometry-based assays such as the intracellular cytokine staining (ICS) strongly improved the capacity to detect several markers on a single cell level. RESULTS: The cumulative analysis of 275 samples from 31 different HIV-1 infected individuals using an ICS staining procedure optimized by our laboratories revealed that, following antigenic stimulation, IFN-gamma producing T-cells were also producing MIP-1beta whereas T-cells characterized by the sole production of IFN-gamma were rare. Since the analysis of the combination of two functions decreases the background and the measurement of the IFN-gamma+ MIP-1beta+ T-cells was equivalent to the measurement of the total IFN-gamma+ T-cells, we adopted the IFN-gamma+ MIP-1beta+ data analysis system to evaluate IFN-gamma-based, antigen-specific T-cell responses. Comparison of our ICS assay with ELISPOT assays performed in two different experienced laboratories demonstrated that the IFN-gamma+ MIP-1beta+ data analysis system increased the sensitivity of the ICS up to levels comparable to the sensitivity of the ELISPOT assay. CONCLUSION: The IFN-gamma+ MIP-1beta+ data evaluation system provides a clear advantage for the detection of low magnitude HIV-1-specific responses. These results are important to guide the choice for suitable highly sensitive immune assays and to build reagent panels able to accurately characterize the phenotype and function of responding T-cells. More importantly, the ICS assay can be used as primary assay to evaluate HIV-1-specific responses without losing sensitivity in comparison to the ELISPOT assay.

Evaluation of modified vaccinia virus Ankara as an alternative vaccine against smallpox in chronically HIV type 1-infected individuals undergoing HAART.

The fear of malevolent use of variola virus by terrorists has led to the implementation of a health care worker vaccination program and to the consideration of vaccination for the general public. However, due to concerns about side effects of the classical smallpox vaccine, especially for immunocompromised individuals, a safer vaccine is urgently needed. We characterized the immunogenicity of modified vaccinia virus Ankara (MVA), one of the more promising alternative smallpox vaccines, in a cohort of 10 chronically HIV-1-infected individuals undergoing highly active antiretroviral therapy (HAART). Nine subjects received smallpox vaccination as children while one subject was never vaccinated against smallpox. All the subjects had CD4 counts >400 cells/mm(3) and 8 out of 10 had undetectable viral loads. MVA was able to elicit humoral and cellular immune responses in the majority of individuals. Vaccinia-specific antibodies were mainly of the IgG class while T cells specific to vaccinia were predominantly CD8(+). The immune responses were maintained over 1 year. Similar vaccinia specific humoral immune responses were observed when our cohort of HIV-1-infected individuals was compared to smallpox-vaccinated healthy subjects. The observed immune responses suggest that the highly attenuated MVA could be used as a substitute vaccine against smallpox in chronically HIV-1-infected individuals undergoing HAART.

Cell line dependent RNA expression profiles of prion-infected mouse neuronal cells.

The overall impact of prion disease on gene expression is not well characterized. We have carried out a large-scale expression analysis of specific cell types commonly employed in studies of prion disease. Neuroblastoma cells (N2a) and hypothalamic neuronal cells (GT1) can be persistently infected with mouse-adapted scrapie prions, the latter demonstrating cytopathologic effects associated with prion neuropathology. Exploiting a mouse DNA microarray containing approximately 21,000 spotted cDNAs, we have identified several hundred differentially expressed sequences in the two cell lines when infected with prion strain RML. ScN2a and ScGT1 cells demonstrate unique changes in RNA profiles and both differ from the reported changes in human microglia and prion-infected brain studies albeit with some overlap. In addition, several of the identified changes are shared in common with other neurodegenerative diseases such as Alzheimer's disease. The results illustrate that prion infection differs in effect depending on cell type, which could be exploited for diagnostic or therapeutic intervention.

Clinical efficacy of melittin in the treatment of cats infected with the feline immunodeficiency virus. / Wirksamkeit von Melittin bei der Behandlung von Katzen mit feliner Immunschwächevirusinfektion.

OBJECTIVE: The bee venom melittin shows an antiviral efficacy against the human immunodeficiency virus in cell culture. It was shown to be non-toxic for cats. Aim of this pilot study was to investigate the clinical efficacy and side-effects of melittin in cats naturally infected with feline immunodeficiency virus (FIV). MATERIAL AND METHODS: The study was performed as a prospective, placebo-controlled double-blinded trial. Twenty cats were included, of which 10 cats each were treated with either melittin (500 µg/kg body weight) or phosphate-buffered saline (placebo) subcutaneously twice per week. During the treatment period of 6 weeks, the cats' general health status, determined by the Karnofsky's score, and the severity of clinical signs (conjunctivitis and stomatitis) using a clinical scoring system were evaluated. Haematology, biochemistry profiles, lymphocyte subpopulations, CD4/CD8 ratio, and pterines (biopterine, 7-xanthopterine) as surrogate parameters were also compared. RESULTS: The general health status and the clinical scores for conjunctivitis and stomatitis improved in cats treated with melittin. A statistically significant improvement however could only be detected for conjunctivitis in cats treated with melittin compared to cats treated with placebo which was likely due to different scores between both groups at the beginning. No influence on the lymphocyte subpopulations, CD4/CD8 ratio, and pterine concentrations was observed. No side effects occurred in this study. CONCLUSION AND CLINICAL RELEVANCE: In the protocol used in the present study, no significant efficacy of melittin could be detected. However, efficacy of melittin, especially if applied in a higher dosage as in the present study or for a longer period, could be evaluated in further studies. Synergistic effects if used in combination with classic antiretroviral drugs could be an interesting future approach.

Identification of a novel Rev-interacting cellular protein.

BACKGROUND: Human cell types respond differently to infection by human immunodeficiency virus (HIV). Defining specific interactions between host cells and viral proteins is essential in understanding how viruses exploit cellular functions and the innate strategies underlying cellular control of HIV replication. The HIV Rev protein is a post-transcriptional inducer of HIV gene expression and an important target for interaction with cellular proteins. Identification of Rev-modulating cellular factors may eventually contribute to the design of novel antiviral therapies. RESULTS: Yeast-two hybrid screening of a T-cell cDNA library with Rev as bait led to isolation of a novel human cDNA product (16.4.1). 16.4.1-containing fusion proteins showed predominant cytoplasmic localization, which was dependent on CRM1-mediated export from the nucleus. Nuclear export activity of 16.4.1 was mapped to a 60 amino acid region and a novel transport signal identified. Interaction of 16.4.1 with Rev in human cells was shown in a mammalian two-hybrid assay and by colocalization of Rev and 16.4.1 in nucleoli, indicating that Rev can recruit 16.4.1 to the nucleus/nucleoli. Rev-dependent reporter expression was inhibited by overexpressing 16.4.1 and stimulated by siRNAs targeted to 16.4.1 sequences, demonstrating that 16.4.1 expression influences the transactivation function of Rev. CONCLUSION: These results suggest that 16.4.1 may act as a modulator of Rev activity. The experimental strategies outlined in this study are applicable to the identification and biological characterization of further novel Rev-interacting cellular factors.

Vaccines based on Nef and on Nef/DeltaV2 Env.

Modified vaccinia virus Ankara (MVA) is a potent vaccine vector, which proved its safety, immunogenity and efficacy in preclinical and clinical studies. The rational for the development of a vaccine against HIV based on the regulatory protein Nef delivered by MVA combined with a V2-deleted Env protein is discussed.

Autologous cytotoxicity of natural killer cells derived from HIV-infected patients.

NK cells recognize target cells with reduced expression of MHC class I molecules. Human immunodeficiency virus (HIV) infection decreases MHC class I on the cell membrane. The aim of this study was to directly evaluate the role and conditions of NK cell effects in HIV seropositive patients ex vivo. Autologous HIV-infected CD4+ T cells were exposed to NK cells recognition. We discovered an increased lysis of the target cells after infection with human immunodeficiency virus-1 (HIV-1). The expression of the HIV-1 nef gene or the combined expression of nef and tat confers NK susceptibility to autologous CD4+ targets. Downregulation of MHC class I but not HLA-C or CD4 correlated with increased recognition by NK cells. The specific recognition is correlated with downregulation of MHC class I molecules on the infected target cells.

Evaluation in rhesus macaques of Tat and rev-targeted immunization as a preventive vaccine against mucosal challenge with SHIV-BX08.

Recent evidence suggests that a CD8-mediated cytotoxic T-cell response against the regulatory proteins of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) may control infection after pathogenic virus challenge. Here, we evaluated whether vaccination with Tat or Tat and Rev could significantly reduce viral load in nonhuman primates. Rhesus macaques were primed with Semliki forest Virus (SFV) expressing HIV-1 tat (SFV-tat) and HIV-1 rev (SFV-rev) and boosted with modified vaccinia virus Ankara (MVA) expressing tat and rev. A second group of monkey was primed with SFV-tat only and boosted with MVA-tat. A third group received a tat and rev DNA/MVA prime-boost vaccine regimen. Monitoring of anti-Tat and anti-Rev antibody responses or antigen-specific IFN-gamma production, as measured by enzyme-linked immunospot assays revealed no clear differences between the three groups. These results suggest that priming with either DNA or SFV seemed to be equivalent, but the additive or synergistic effect of a rev vaccine could not be clearly established. The animals were challenged by the rectal route 9 weeks after the last booster immunization, using 10 MID(50) of a SHIV-BX08 stock. Postchallenge follow-up of the monkeys included testing seroconversion to Gag and Env antigens, measuring virus infectivity in PBMC by cocultivation with noninfected human cells, and monitoring of plasma viral load. None of the animals was protected from infection as assessed by PCR, but peak viremia was reduced more than 200-fold compared to sham controls in one third (6/18) of vaccinated macaques, whatever the vaccine regimen they received. Interestingly, among these six protected animals four did not seroconvert. Altogether, these results clearly indicated that the addition of early HIV proteins like Tat and Rev in a multicomponent preventive vaccine including structural proteins like Env or Gag may be beneficial in preventive vaccinal strategies.

Generation of recombinant fowlpox virus using the non-essential F11L orthologue as insertion site and a rapid transient selection strategy.

Avipoxviruses show an abortive replication phenotype in mammalian cells and are under evaluation as safe vectors for vaccination. Non-essential gene sequences located in highly conserved regions of virus genomes are considered particularly useful to integrate heterologous DNA. Fowlpox virus F11L orthologue is described in this paper as a suitable locus for insertion into fowlpox virus genome. Disruption of the F11L coding sequence by integration of an expression cassette for the Escherichia coli lacZ and guanine phosphoribosyltransferase marker genes resulted in the isolation of replication competent knockout viruses. Growth of F11L-knockout viruses in primary chicken embryo fibroblasts was unimpaired in comparison to wild type-virus. To test the generation of vector viruses, an insertion plasmid was constructed that contains F11L-specific sequences for homologous recombination, the E. coli lacZ and gpt genes as transient selectable marker, and the vaccinia virus early/late promoter P7.5 for transcriptional control of target gene expression. The coding sequence of the melanoma-associated antigen tyrosinase was chosen as model recombinant gene. Isolation of tyrosinase-recombinant viruses, which produced stably the insert, demonstrated the usefulness of the F11L-insertion site for the generation of fowlpox vectors. Rapid isolation of those recombinants was achieved by using a double selective system and linearising the vector plasmid before transfection.

Gene Expression Driven by a Strong Viral Promoter in MVA Increases Vaccination Efficiency by Enhancing Antibody Responses and Unmasking CD8⁺ T Cell Epitopes.

Viral vectors are promising tools for vaccination strategies and immunotherapies. However, CD8⁺ T cell responses against pathogen-derived epitopes are usually limited to dominant epitopes and antibody responses to recombinant encoded antigens (Ags) are mostly weak. We have previously demonstrated that the timing of viral Ag expression in infected professional Ag-presenting cells strongly shapes the epitope immunodominance hierarchy. T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses. In this work we evaluate the effect of overexpressing the recombinant Ag using the modified vaccinia virus early/late promoter H5 (mPH5). Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced. The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags. Moreover, the increase in Ag-secretion favors the induction of strong antibody responses. Our findings provide the rationale to develop new strategies for fine-tuning the responses elicited by recombinant modified vaccinia virus Ankara by using selected promoters to improve the performance of this viral vector.

Longitudinal changes in HIV-1-specific T-cell quality associated with viral load dynamic.

BACKGROUND: Several correlates of HIV control have been described; however their predictive values remain unclear, since most studies have been performed in cross-sectional settings. OBJECTIVES: We evaluated the cause and consequence relationship between quality of HIV-specific T-cell response and viral load dynamic in a temporal perspective. STUDY DESIGN: HIV-1-specific T-cell responses were monitored over 7 years in a patient that following treatment interruption maintained a stable/low viral set point for 3.1 years before control of viral replication was lost and antiretroviral therapy restarted. RESULTS: We observed that high frequencies of HIV-1-specific CD4 and CD8 T cells were unable to prevent loss of viral control. Gradual loss of functionality was observed in these responses, characterized by early loss of IL-2, viral load-dependent decrease of IFN-γ and CD154 expression as well as increase of MIP-1ß production. Terminally differentiated HIV-1-specific CD8 T cells expressing CD45RA were lost independently of viral load and preceded the loss-of-control phase of HIV infection. CONCLUSION: By describing qualitative changes in HIV-1-specific T-cell responses that coincide with loss of viral control, we identified specific correlates of disease progression and putative markers of viral control. Our findings suggest including the markers IL-2, IFN-γ, MIP-1ß, CD154 and CD45RA into monitoring of HIV-specific T-cell-responses to prospectively determine correlates of protection from disease-progression.

A new antigen scanning strategy for monitoring HIV-1 specific T-cell immune responses.

Delineation of the immune correlates of protection in natural infection or after vaccination is a mandatory step for vaccine development. Although the most recent techniques allow a sensitive and specific detection of the cellular immune response, a consensus on the best strategy to assess their magnitude and breadth is yet to be reached. Within the AIDS Vaccine Integrated Project (AVIP http://www.avip-eu.org) we developed an antigen scanning strategy combining the empirical-based approach of overlapping peptides with a vast array of database information. This new system, termed Variable Overlapping Peptide Scanning Design (VOPSD), was used for preparing two peptide sets encompassing the candidate HIV-1 vaccine antigens Tat and Nef. Validation of the VOPSD strategy was obtained by direct comparison with 15mer or 20mer peptide sets in a trial involving six laboratories of the AVIP consortium. Cross-reactive background responses were measured in 80 HIV seronegative donors (HIV-), while sensitivity and magnitude of Tat and Nef-specific T-cell responses were assessed on 90 HIV+ individuals. In HIV-, VOPSD peptides generated background responses comparable with those of the standard sets. In HIV-1+ individuals the VOPSD pools showed a higher sensitivity in detecting individual responses (Tat VOPSD vs. Tat 15mers or 20mers: p≤0.01) as well as in generating stronger responses (Nef VOPSD vs. Nef 20mers: p<0.001) than standard sets, enhancing both CD4 and CD8 T-cell responses. Moreover, this peptide design allowed a marked reduction of the peptides number, representing a powerful tool for investigating novel HIV-1 candidate vaccine antigens in cohorts of HIV-seronegative and seropositive individuals.

Modified vaccinia virus Ankara exerts potent immune modulatory activities in a murine model.

BACKGROUND: Modified vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus, has been used as vaccine delivery vector in preclinical and clinical studies against infectious diseases and malignancies. Here, we investigated whether an MVA which does not encode any antigen (Ag) could be exploited as adjuvant per se. METHODOLOGY/PRINCIPAL FINDINGS: We showed that dendritic cells infected in vitro with non-recombinant (nr) MVA expressed maturation and activation markers and were able to efficiently present exogenously pulsed Ag to T cells. In contrast to the dominant T helper (Th) 1 biased responses elicited against Ags produced by recombinant MVA vectors, the use of nrMVA as adjuvant for the co-administered soluble Ags resulted in a long lasting mixed Th1/Th2 responses. CONCLUSIONS/SIGNIFICANCE: These findings open new ways to potentiate and modulate the immune responses to vaccine Ags depending on whether they are co-administered with MVA or encoded by recombinant viruses.

The combination of marker gene swapping and fluorescence-activated cell sorting improves the efficiency of recombinant modified vaccinia virus Ankara vaccine production for human use.

Modified vaccinia virus Ankara (MVA) is employed as a human vaccine vector for the high expression of heterologous genes and the lack of replication in mammalian cells. This study demonstrates that cells infected by recombinant viruses can be obtained by fluorescence-activated cell sorting. Recombinant viruses are generated by a swapping event between a red fluorescent protein gene in the acceptor virus and a plasmid cassette coding for both a green fluorescent marker and a transgene. To prevent the carry-over of parental virus, due to superinfection of the cells harbouring recombinant viruses, the sorting is performed on cells infected at low m.o.i. in the presence of a reversible inhibitor of viral particle release. Terminal dilution cloning is then used to isolate both green and marker-free recombinant viruses, which can be identified by whole-plate fluoroimaging. The differential visualization of all the viral types involved allows a stepwise monitoring of all recombinations and leads to a straightforward and efficient flow cytometry-based cell sorting purification protocol. As an example of the efficacy of this sorting procedure, the construction of rMVA's coding for the rat nuclear protein HMGB1 and H5N1 influenza A virus hemagglutinin is reported. The entire recombinant MVA production process is carried out in serum-free media employing primary chicken embryo fibroblasts (CEF), which are certified for the preparation of human vaccines. This rMVA production method is faster, simpler and more reliable than any other available procedure for obtaining safe vaccine stocks for human use.