Safety and Immunogenicity of Novel Recombinant BCG and Modified Vaccinia Virus Ankara Vaccines in Neonate Rhesus Macaques

Although major inroads into making antiretroviral therapy available in resource-poor countries have been made, there is an urgent need for an effective vaccine administered shortly after birth, which would protect infants from acquiring human immunodeficiency virus type 1 (HIV-1) through breast-feeding. Bacillus Calmette-Guérin (BCG) is given to most infants at birth, and its recombinant form could be used to prime HIV-1-specific responses for a later boost by heterologous vectors delivering the same HIV-1-derived immunogen. Here, two groups of neonate Indian rhesus macaques were immunized with either novel candidate vaccine BCG.HIVA⁴⁰¹ or its parental strain AERAS-401, followed by two doses of recombinant modified vaccinia virus Ankara MVA.HIVA. The HIVA immunogen is derived from African clade A HIV-1. All vaccines were safe, giving local reactions consistent with the expected response at the injection site. No systemic adverse events or gross abnormality was seen at necropsy. Both AERAS-401 and BCG.HIVA⁴⁰¹ induced high frequencies of BCG-specific IFN-γ-secreting lymphocytes that declined over 23 weeks, but the latter failed to induce detectable HIV-1-specific IFN-γ responses. MVA.HIVA elicited HIV-1-specific IFN-γ responses in all eight animals, but, except for one animal, these responses were weak. The HIV-1-specific responses induced in infants were lower compared to historic data generated by the two HIVA vaccines in adult animals but similar to other recombinant poxviruses tested in this model. This is the first time these vaccines were tested in newborn monkeys. These results inform further infant vaccine development and provide comparative data for two human infant vaccine trials of MVA.HIVA.Close to 2.3 million of children globally are infected with human immunodeficiency virus type 1 (HIV-1). The majority of neonatal infections occur in utero or intrapartum and, in the absence of preventative interventions, up to 29% of infants breast-fed by infected mothers acquire HIV-1 (6). Furthermore, HIV-1-infected children face a worse prognosis than adults in that, without antiretroviral treatment (ART), 25% of perinatally infected children progress to AIDS within 1 year (10), and the median time to AIDS for the remaining children is less than 7 years (2). It is now clearly established that maternal and extended infant ART can substantially reduce transmission of HIV-1 through breast-feeding (23). However, in a resource-poor setting, many logistical barriers to implementation of the ART-based prevention of mother-to-child-transmission (PMTCT) remain (23). Because nutrition and hygiene makes breast milk an important determinant of infant survival (22, 28), formula feeding as a protective measure against HIV-1 acquisition is recommended only if it is AFASS (acceptable, feasible, affordable, sustainable, and safe). Unfortunately, AFASS it is still not for majority of infected mothers in sub-Saharan Africa. Also, mixed bottle and breast feeding is associated with a 10-fold increase in HIV-1 transmission relative to exclusive breast-feeding (4). Thus, an effective infant vaccine against HIV-1 infection is the best and safest solution for PMTCT of HIV-1 with the added practical option of prolonging breast-feeding.Neonatal immunity is immature compared to the adult immune system (25). The differences include naivety of the immune cells, a tendency to develop Th2 responses (5) and antigen-presenting cells with inefficient cytokine production (35). For example, human cord blood T cells proliferated poorly and produced low levels of interleukin-2 (IL-2) and gamma interferon (IFN-γ) when endogenous antigen-presenting cells presented the antigen (35, 44). Also, infant myeloid dendritic cells are less efficient in priming Th1 responses because of their decreased responsiveness to Toll-like receptor stimulation, lower levels of surface costimulatory molecules, and lower production of IL-12 (8, 27). In several infections, qualitative and quantitative differences between human newborn and adult responses were detected (1, 9, 26, 37). In contrast, other studies of infants reported proliferation as well as IL-2 and IFN-γ production by T cells equal to that of adults following T-cell receptor-independent activation (21, 46). These latter observations indicate that neonate T cells are not intrinsically “locked” into an immature phenotype but, given the correct stimuli, they can develop mature immune responses (25). The requirement for specific stimuli will likely differ for different pathogens and vaccine vectors.Mycobacterium bovis bacillus Calmette-Guérin (BCG) is commonly delivered at birth as an antituberculosis vaccine as a part of the WHO Expanded Programme on Immunization (EPI). It has been reported by several studies to promote an adultlike Th1 response in newborns (16, 24, 34, 43), although it was also suggested that delaying the BCG delivery to 10 weeks of age benefits the quantity and quality of BCG-induced CD4 T-cell responses (20). BCG and related mycobacterial vectors have been explored as vaccines against other infectious agents, including human and simian immunodeficiency viruses (19), and in adult animals showed immunogenicity and protection (3, 36, 39, 47, 48). The only clinical study of recombinant BCG (rBCG) in adults failed to provide consistent efficacy (7). We have suggested the use of rBCG expressing an HIV-1-derived immunogen as the priming component of a heterologous vaccine platform for PMTCT of HIV-1 through infected breast milk (18), where it is critical to prime HIV-1-specific responses as soon as possible after birth. These responses could be boosted a few weeks later or shortly after the already busy EPI by heterologous vaccines delivering the same HIV-1-derived immunogen. To this extent, we constructed the novel candidate vaccine BCG.HIVA⁴⁰¹ (36) by inserting a gene coding for the HIV-1 clade A-derived immunogen HIVA (14) into recombinant BCG strain AREAS-401 (40). AERAS-401 is a newly developed strain that displayed enhanced safety (40) and immunogenicity (11, 15) in murine models relative to its parental BCG vaccine strain Danish SSI-1331. Increased safety represents an important feature should the BCG.HIVA⁴⁰¹ vaccine be deployed in babies born to HIV-1-infected mothers. We showed that BCG.HIVA⁴⁰¹ in a heterologous combination with recombinant modified vaccinia virus Ankara MVA.HIVA and recombinant ovine atadenovirus OAdV.HIVA induced robust polyfunctional HIV-1-specific T-cell responses in adult macaques (36). Here, we assess the safety and immunogenicity of the BCG.HIVA prime-MVA.HIVA boost regimen in newborn rhesus macaques.     

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.     

Oat Bran as a Cholesterol-Reducing Dietary Adjunct in a Young,Healthy Population

In a study to examine the effects of dietary oat bran on serum lipids, subjects who ate two oat bran muffins a day for 28 days showed a 5.3% reduction in serum total cholesterol and an 8.7% reduction in low-density-lipoprotein-cholesterol levels, while no changes were noted in subjects consuming wheat or mixed wheat and oat bran muffins. Similarly, those who ingested oat bran muffins showed an 8.3% reduction in serum triglyceride values as contrasted with an overall increase of 6.4% in the other groups combined. These findings suggest that oat bran taken daily can significantly lower serum cholesterol and triglyceride levels in a young, healthy population.     

Recombinant Modified Vaccinia Virus Ankara Expressing Glycoprotein E2 of Chikungunya Virus Protects AG129 Mice against Lethal Challenge

Chikungunya virus (CHIKV) infection is characterized by rash, acute high fever, chills, headache, nausea, photophobia, vomiting, and severe polyarthralgia. There is evidence that arthralgia can persist for years and result in long-term discomfort. Neurologic disease with fatal outcome has been documented, although at low incidences. The CHIKV RNA genome encodes five structural proteins (C, E1, E2, E3 and 6K). The E1 spike protein drives the fusion process within the cytoplasm, while the E2 protein is believed to interact with cellular receptors and therefore most probably constitutes the target of neutralizing antibodies. We have constructed recombinant Modified Vaccinia Ankara (MVA) expressing E3E2, 6KE1, or the entire CHIKV envelope polyprotein cassette E3E26KE1. MVA is an appropriate platform because of its demonstrated clinical safety and its suitability for expression of various heterologous proteins. After completing the immunization scheme, animals were challenged with CHIV-S27. Immunization of AG129 mice with MVAs expressing E2 or E3E26KE1 elicited neutralizing antibodies in all animals and provided 100% protection against lethal disease. In contrast, 75% of the animals immunized with 6KE1 were protected against lethal infection. In conclusion, MVA expressing the glycoprotein E2 of CHIKV represents as an immunogenic and effective candidate vaccine against CHIKV infections.     

修饰的痘苗病毒安卡拉株（MVA）基因组中高频的同源重组

痘苗病毒由于其外源基因容量大,表达产物后加工完善等优势而广泛用于基因工程的研究以及基因治疗,痘苗病毒基因组的同源重组现象为其基因操作带来了方便,也被用于很多痘苗病毒基因结构和功能的研究,痘苗病毒安卡拉株（MVA）是一种修饰的复制限制的痘苗病毒,由于极高的安全性,正在实验室和临床应用的很多领域取代普通的痘苗病毒,为提高重组MVA系统的安全性以及筛选重组MVA的效率,发展了一种暂时选择系统,此系统利用分子内2段同向的相同序列发生同源重组去除选择标记k1l基因,从而消除选择标记对宿主可能的危害。利用此暂时表达系统构建了4个携带编码不同长度外源多蛋白质序列的重组MVA,并估算了每次传代的重组频率,结果显示,MVA同源重组频率虽然比其他痘苗病毒株要低,但仍然是较斋的,将带有k1l基因的重组MVA经3-4次盲传（blind passage),即可获得完全去除选择标记的重组MVA。进一步证明上述利用暂时选择标记k1l基因构建重组MVA的系统具有十分可靠的安全性,适合作为人体活疫苗开发和基因治疗的载体,而且,通过盲传进行筛选,能大大提高去除选择标记的效率,降低鸺建重组MVA的成本。     

A Prospective Study of the Incidence of Myocarditis/Pericarditis and New Onset Cardiac Symptoms following Smallpox and Influenza Vaccination

Background

Although myocarditis/pericarditis (MP) has been identified as an adverse event following smallpox vaccine (SPX), the prospective incidence of this reaction and new onset cardiac symptoms, including possible subclinical injury, has not been prospectively defined.

Purpose

The study’s primary objective was to determine the prospective incidence of new onset cardiac symptoms, clinical and possible subclinical MP in temporal association with immunization.

Methods

New onset cardiac symptoms, clinical MP and cardiac specific troponin T (cTnT) elevations following SPX (above individual baseline values) were measured in a multi-center prospective, active surveillance cohort study of healthy subjects receiving either smallpox vaccine or trivalent influenza vaccine (TIV).

Results

New onset chest pain, dyspnea, and/or palpitations occurred in 10.6% of SPX-vaccinees and 2.6% of TIV-vaccinees within 30 days of immunization (relative risk (RR) 4.0, 95% CI: 1.7-9.3). Among the 1081 SPX-vaccinees with complete follow-up, 4 Caucasian males were diagnosed with probable myocarditis and 1 female with suspected pericarditis. This indicates a post-SPX incidence rate more than 200-times higher than the pre-SPX background population surveillance rate of myocarditis/pericarditis (RR 214, 95% CI 65-558). Additionally, 31 SPX-vaccinees without specific cardiac symptoms were found to have over 2-fold increases in cTnT (>99th percentile) from baseline (pre-SPX) during the window of risk for clinical myocarditis/pericarditis and meeting a proposed case definition for possible subclinical myocarditis. This rate is 60-times higher than the incidence rate of overt clinical cases. No clinical or possible subclinical myocarditis cases were identified in the TIV-vaccinated group.

Conclusions

Passive surveillance significantly underestimates the true incidence of myocarditis/pericarditis after smallpox immunization. Evidence of subclinical transient cardiac muscle injury post-vaccinia immunization is a finding that requires further study to include long-term outcomes surveillance. Active safety surveillance is needed to identify adverse events that are not well understood or previously recognized.     

Effective Induction of Simian Immunodeficiency Virus-Specific Cytotoxic T Lymphocytes in Macaques by Using a Multiepitope Gene and DNA Prime-Modified Vaccinia Virus Ankara Boost Vaccination Regimen

DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8(+) lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed.     

Safety and Tolerability of Conserved Region Vaccines Vectored by Plasmid DNA,Simian Adenovirus and Modified Vaccinia Virus Ankara Administered to Human Immunodeficiency Virus Type 1-Uninfected Adults in a Randomized,Single-Blind Phase I Trial

Trial Design

HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee) adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported.

Methods

Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination.

Results

Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1) and predominantly transient (<48 hours). Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range) of 633 (231-1533) post-vaccination, which is of no safety concern.

Conclusions

These data demonstrate safety and good tolerability of the pSG2.HIVconsv DNA, ChAdV63.HIVconsv and MVA.HIVconsv vaccines and together with their high immunogenicity support their further development towards efficacy studies.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01151319","term_id":"NCT01151319"}}NCT01151319     

Phase 1 Safety and Immunogenicity Evaluation of ADMVA,a Multigenic,Modified Vaccinia Ankara-HIV-1 B'/C Candidate Vaccine

Background

We conducted a Phase I dose-escalation trial of ADMVA, a Clade-B''/C-based HIV-1 candidate vaccine expressing env, gag, pol, nef, and tat in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the safety and immunogenicity of ADMVA in human volunteers.

Methodology/Principal Findings

ADMVA or placebo was administered intramuscularly at months 0, 1 and 6 to 50 healthy adult volunteers not at high risk for HIV-1. In each dosage group [1×10⁷ (low), 5×10⁷ (mid), or 2.5×10⁸ pfu (high)] volunteers were randomized in a 3∶1 ratio to receive ADMVA or placebo in a double-blinded design. Subjects were followed for local and systemic reactogenicity, adverse events including cardiac adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA, immunoflourescent staining, and HIV-1 neutralization. Cellular immunogenicity was assessed by a validated IFNγ ELISpot assay and intracellular cytokine staining. Anti-vaccinia binding titers were measured by ELISA.ADMVA was generally well-tolerated, with no vaccine-related serious adverse events or cardiac adverse events. Local or systemic reactogenicity events were reported by 77% and 78% of volunteers, respectively. The majority of events were of mild intensity. The IFNγ ELISpot response rate to any HIV antigen was 0/12 (0%) in the placebo group, 3/12 (25%) in the low dosage group, 6/12 (50%) in the mid dosage group, and 8/13 (62%) in the high dosage group. Responses were often multigenic and occasionally persisted up to one year post vaccination. Antibodies to gp120 were detected in 0/12 (0%), 8/13 (62%), 6/12 (50%) and 10/13 (77%) in the placebo, low, mid, and high dosage groups, respectively. Antibodies persisted up to 12 months after vaccination, with a trend toward agreement with the ability to neutralize HIV-1 SF162 in vitro. Two volunteers mounted antibodies that were able to neutralize clade-matched viruses.

Conclusions/Significance

ADMVA was well-tolerated and elicited durable humoral and cellular immune responses.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00252148","term_id":"NCT00252148"}}NCT00252148     

Predictors of Three Dose On-Time Compliance with HPV4 Vaccination in a Disadvantaged,Underserved, Safety Net Population in the US Midwest

Background

HPV4 is approved as a series of three timed doses expected to result in efficacy against specific HPV infections. Completion rates in the US are quite low at the same time the structure of health care delivery is changing. The aim of this study was to determine how the patient-, clinic- and systems-level characteristics facilitate or hinder the timely completion of three HPV4 doses in both adolescent and adult female populations in a high-risk safety net population.

Methods

This is a retrospective study in which patient-, clinic- and systems-level data are abstracted from the electronic medical record (EMR) for all females 10–26 years of age receiving at least one dose of HPV4 between July 1, 2006 and October 1, 2009.

Results

Adults were more likely to complete the three dose series if they had at least one health care visit in addition to their HPV4 visit, (aOR = 1.54 (95% CI:1.10, 2.15). Adults were less likely to complete the three dose series if they received their second HPV4 dose at an acute health care, preventive care or postpartum visits compared to an HPV4-only visit (aOR = 0.31 (95% CI: 0.13, 0.72), 0.12 (0.04, 0.35), 0.30 (0.14, 0.62), respectively). Hispanic adults were less likely than whites to complete the series (aOR = 0.24 (95% CI:0.10, 0.59). 39% of adolescents who completed two doses completed the series.

Conclusions

HPV4 is more likely to be effectively administered to adults in a safety net population if multiple health care needs can be met within the health care system.     

Use of a Recombinant Vaccinia Virus Expressing Interferon Gamma for Post-Exposure Protection against Vaccinia and Ectromelia Viruses

Post-exposure vaccination with vaccinia virus (VACV) has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. While there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. In this study, we analyzed post-exposure prophylaxis using several attenuated recombinant VACV in a mouse model. A recombinant VACV expressing murine interferon gamma (IFN-γ) was most effective for post-exposure protection of mice infected with VACV and ectromelia virus (ECTV). Untreated animals infected with VACV exhibited severe weight loss and morbidity leading to 100% mortality by 8 to 10 days post-infection. Animals treated one day post-infection had milder symptoms, decreased weight loss and morbidity, and 100% survival. Treatment on days 2 or 3 post-infection resulted in 40% and 20% survival, respectively. Similar results were seen in ECTV-infected mice. Despite the differences in survival rates in the VACV model, the viral load was similar in both treated and untreated mice while treated mice displayed a high level of IFN-γ in the serum. These results suggest that protection provided by IFN-γ expressed by VACV may be mediated by its immunoregulatory activities rather than its antiviral effects. These results highlight the importance of IFN-γ as a modulator of the immune response for post-exposure prophylaxis and could be used potentially as another post-exposure prophylaxis tool to prevent morbidity following infection with smallpox and other orthopoxviruses.     

Disseminated Candidiasis in a Young,Previously Healthy,Dog and Review of Literature

Background

The reports on disseminated candidiasis in dogs so far describe at least one predisposing factor. This case report, however, highlights candidiasis in a dog without any known predisposition.

Patient

A 1.5-year-old intact female Hovawart dog was presented with subcutaneous nodules and polyuria/polydipsia. An excisional biopsy revealed a chronic pyogranulomatous and necrotizing inflammation with mycotic structures. The patient became febrile and lethargic, and developed lameness.

Methods

A physical examination, blood tests, urinalysis, thoracic radiographs, abdominal ultrasonography of the abdomen, fine-needle aspiration biopsies, and a culture of a subcutaneous nodule aspirate were obtained. Selected sections of multiple organs were collected for routine histology postmortem. The isolate and a subcutaneous mass were subjected to molecular identification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS) analysis.

Results

Clinical, laboratory, and radiological findings were consistent with a granulomatous chronic systemic inflammation. Cytology and histology showed a pyogranulomatous and necrotizing inflammation with myriads of intra- and extra-cellular yeasts and extracellular hyphae. Culture yielded numerous yeast colonies, which appeared Candida albicans–like, but showed a negative serum test and a low identification in API 20 C AUX. Nucleic acid sequences showed homology with the C. albicans-type strain CBS 562. Multilocus sequence typing (MLST) resulted in a new type with designation DST121. The identification of the isolates was confirmed by MALDI-TOF–MS analysis.

Conclusion and Clinical Importance

Future MLST typing and investigation of virulence can provide further evidence whether this MLST-type is associated with clinical cases of disseminated candidiasis without an apparent predisposing condition.

     

Introduction of the Six Major Genomic Deletions of Modified Vaccinia Virus Ankara (MVA) into the Parental Vaccinia Virus Is Not Sufficient To Reproduce an MVA-Like Phenotype in Cell Culture and in Mice

Modified vaccinia virus Ankara (MVA) has a highly restricted host range in cell culture and is apathogenic in vivo. MVA was derived from the parental chorioallantois vaccinia virus Ankara (CVA) by more than 570 passages in chicken embryo fibroblast (CEF) cells. During CEF cell passaging, six major deletions comprising 24,668 nucleotides occurred in the CVA genome. We have cloned both the MVA and the parental CVA genome as bacterial artificial chromosomes (BACs) and have sequentially introduced the six major MVA deletions into the cloned CVA genome. Reconstituted mutant CVA viruses containing up to six major MVA deletions showed no detectable replication restriction in 12 of 14 mammalian cell lines tested; the exceptions were rabbit cell lines RK13 and SIRC. In mice, CVA mutants with up to three deletions showed slightly enhanced virulence, suggesting that gene deletion in replicating vaccinia virus (VACV) can result in gain of fitness in vivo. CVA mutants containing five or all six deletions were still pathogenic, with a moderate degree of attenuation. Deletion V was mainly responsible for the attenuated phenotype of these mutants. In conclusion, loss or truncation of all 31 open reading frames in the six major deletions is not sufficient to reproduce the specific MVA phenotype of strong attenuation and highly restricted host range. Mutations in viral genes outside or in association with the six major deletions appear to contribute significantly to this phenotype. Host range restriction and avirulence of MVA are most likely a cooperative effect of gene deletions and mutations involving the major deletions.Modified vaccinia virus Ankara (MVA) was derived from the parental strain chorioallantois vaccinia virus Ankara (CVA) by more than 570 passages in chicken embryo fibroblast (CEF) cells and became severely host cell restricted to avian cells (6, 9, 26). MVA is apathogenic in mammalian hosts, while maintaining excellent immunogenicity (5, 18, 41, 46). Due to the versatility of MVA as a gene expression vector and its immunogenicity, MVA offers an attractive basis for recombinant vector vaccines (17, 48, 50). In addition, the recent appreciation of the possibility of accidental or deliberate release of the smallpox virus renewed interest in an MVA-based smallpox vaccine. MVA was used in the 1970s as a priming vaccine prior to the administration of conventional smallpox vaccine in a two-step program. No significant adverse events were reported after the administration of MVA to more than 120,000 primary vaccinees in Germany (27, 45). Recent clinical studies using a third-generation vaccine, MVA-BN, as a stand-alone smallpox vaccine confirmed its excellent safety profile (56, 57) and underscored its potential as a safe vaccine vector against human infections with various pathogens as well as against cancer.One important reason for the versatility of MVA as a vaccine vector is its particular host range phenotype, which allows the viral gene expression program to proceed until late times in infection, resulting in efficient expression of viral as well as recombinant proteins. The block in viral replication occurs very late during assembly of mature and infectious viral particles (42, 48). With the notable exception of the Syrian hamster cell line BHK-21 and the recently described rat cell line IEC-6, MVA has a very limited ability to productively replicate in mammalian cells (9, 16, 35). The genetic basis of the particular host range restriction of MVA is still not well defined. Comparisons with NYVAC, another replication-deficient vaccinia virus vector (54), showed that significant differences in gene expression programs, apoptosis induction, and immunogenicity exist between NYVAC and MVA (19, 31, 32), although they have very similar host ranges in vitro. Thus, comprehensive knowledge of the genetic factors determining the host ranges of such vectors is necessary to provide a deeper understanding of the basis for the safety and immunogenicity to eventually allow their further optimization.In the course of passaging CVA on CEF cells, the virus acquired six large genomic deletions totaling more than 24 kbp of genomic DNA and deleting or truncating 31 open reading frames (ORFs). In addition to the six major deletions, a multitude of shorter deletions and insertions as well as point mutations have occurred in the MVA genome, resulting in gene fragmentation, truncation, short internal deletions, and amino acid exchanges (29). Some or all of these mutations might also contribute to the attenuated phenotype of MVA. MVA no longer encodes many of the known poxviral immune evasion and virulence factors (2). Of the five classical host range genes present in vaccinia virus (VACV), only C12L/SPI-1 and K1L are deleted or truncated in MVA, whereas C7L, K3L, and E3L are preserved. Deletion of C12L/SPI-1 and K1L contributed to the limited MVA host range, but their reconstitution only partially reversed the MVA host range restriction in selected cell lines (49, 59). Marker rescue experiments using large fragments of the CVA genome suggested that at least two further host range genes apart from C12L/SPI-1, C7L, and K1L might reside in the left part of the VACV genome (59).It is presently unknown how the multiple genetic alterations of MVA determine its limited ability to replicate in most mammalian cells and its lack of pathogenicity in vivo. Since restricted host range and lack of pathogenicity of MVA have commonly been associated with the large deletions in the MVA genome, we aimed at sequentially introducing the six major MVA-like deletions in CVA. To facilitate and accelerate mutagenesis, we have generated bacterial artificial chromosome (BAC) clones of both the MVA and CVA genomes. The use of a counterselectable marker allowed multiple consecutive rounds of mutagenesis of the CVA-BAC (39, 58, 62). The resulting CVA mutants showed that even the introduction of all six major MVA deletions did not create an MVA-like host range phenotype and caused only slight attenuation of the parental CVA virus in a murine intranasal infection model. This result indicates that major host range determinants of MVA are located outside the six large deletions. The host range and virulence phenotype of MVA most probably result from a combined effect of mutation of these unknown factors in conjunction with the six major deletions.