Priming with Secreted Glycoprotein G of Respiratory Syncytial Virus (RSV) Augments Interleukin-5 Production and Tissue Eosinophilia after RSV Challenge

The respiratory syncytial virus (RSV) G glycoprotein promotes differentiation of type 2 CD4⁺ T lymphocytes and induces an eosinophilic response in lungs of RSV-infected mice. A unique feature of G is that a second initiation codon in the transmembrane region of the glycoprotein results in secretion of soluble protein from infected cells. Recombinant vaccinia viruses that express wild-type G (vvWT G), only secreted G (vvM48), or only membrane-anchored G (vvM48I) were used to define the influence of G priming on immunopathogenesis. Mice immunized with vvM48 had more severe illness following RSV challenge than did mice primed with vvWT G or vvM48I. Coadministration of purified G during priming with the construct expressing membrane-anchored G shifted immune responses following RSV challenge to a more Th2-like response. This was characterized by increased interleukin-5 in lung supernatants and an increase in G-specific immunoglobulin G1 antibodies. Eosinophils were present in the infiltrate of all mice primed with G-containing vectors but were greatest in mice primed with regimens including secreted G. These data suggest the form of G protein available for initial antigen processing and presentation is an important factor in promoting Th2-like immune responses, including the induction of lung eosinophilia. The ability of RSV to secrete G protein may therefore represent a viral strategy for immunomodulation and be a key determinant of disease pathogenesis.     

Recombinant Respiratory Syncytial Virus (RSV) Bearing a Set of Mutations from Cold-Passaged RSV Is Attenuated in Chimpanzees

A set of five missense mutations previously identified by nucleotide sequence analysis of subgroup A cold-passaged (cp) respiratory syncytial virus (RSV) has been introduced into a recombinant wild-type strain of RSV. This recombinant virus, designated rA2cp, appears to replicate less efficiently in the upper and lower respiratory tracts of seronegative chimpanzees than either biologically derived or recombinant wild-type RSV. Infection with rA2cp also resulted in significantly less rhinorrhea and cough than infection with wild-type RSV. These findings confirm the role of the cp mutations in attenuation of RSV and identify their usefulness for inclusion in future live attenuated recombinant RSV vaccine candidates.     

Kinetics of Respiratory Syncytial Virus (RSV) Memphis Strain 37 (M37) Infection in the Respiratory Tract of Newborn Lambs as an RSV Infection Model for Human Infants

Rationale

Respiratory syncytial virus (RSV) infection in preterm and newborn infants can result in severe bronchiolitis and hospitalization. The lamb lung has several key features conducive to modeling RSV infection in human infants, including susceptibility to human strains of RSV such as the A2, Long, and Memphis Strain 37 (M37). In this study, the kinetics of M37 infection was investigated in newborn lambs in order to better define clinical, viral, physiological, and immunological parameters as well as the pathology and lesions.

Methods

Newborn lambs were nebulized with M37 hRSV (6 mL of 1.27 x 10⁷ FFU/mL), monitored daily for clinical responses, and respiratory tissues were collected from groups of lambs at days 1, 3, 4, 6, and 8 post-inoculation for the assessment of viral replication parameters, lesions and also cellular, immunologic and inflammatory responses.

Results

Lambs had increased expiratory effort (forced expiration) at days 4, 6, and 8 post-inoculation. Nasal wash lacked RSV titers at day 1, but titers were present at low levels at days 3 (peak), 4, and 8. Viral titers in bronchoalveolar lavage fluid (BALF) reached a plateau at day 3 (4.6 Log₁₀ FFU/mL), which was maintained until day 6 (4.83 Log₁₀ FFU/mL), and were markedly reduced or absent at day 8. Viral RNA levels (detected by RT-qPCR) in BALF were indistinguishable at days 3 (6.22 ± 0.08 Log₁₀ M37 RNA copies/mL; mean ± se) and 4 (6.20 ± 0.16 Log₁₀ M37 RNA copies/mL; mean ± se) and increased slightly on day 6 (7.15 ± 0.2 Log₁₀ M37 RNA copies/mL; mean ± se). Viral antigen in lung tissue as detected by immunohistochemistry was not seen at day 1, was present at days 3 and 4 before reaching a peak by day 6, and was markedly reduced by day 8. Viral antigen was mainly present in airways (bronchi, bronchioles) at day 3 and was increasingly present in alveolar cells at days 4 and 6, with reduction at day 8. Histopathologic lesions such as bronchitis/bronchiolitis, epithelial necrosis and hyperplasia, peribronchial lymphocyte infiltration, and syncytial cells, were consistent with those described previously for lambs and infants.

Conclusion

This work demonstrates that M37 hRSV replication in the lower airways of newborn lambs is robust with peak replication on day 3 and sustained until day 6. These findings, along with the similarities of lamb lung to those of infants in terms of alveolar development, airway branching and epithelium, susceptibility to human RSV strains, lesion characteristics (bronchiolitis), lung size, clinical parameters, and immunity, further establish the neonatal lamb as a model with key features that mimic RSV infection in infants.     

Latitudinal Variations in Seasonal Activity of Influenza and Respiratory Syncytial Virus (RSV): A Global Comparative Review

Background

There is limited information on influenza and respiratory syncytial virus (RSV) seasonal patterns in tropical areas, although there is renewed interest in understanding the seasonal drivers of respiratory viruses.

Methods

We review geographic variations in seasonality of laboratory-confirmed influenza and RSV epidemics in 137 global locations based on literature review and electronic sources. We assessed peak timing and epidemic duration and explored their association with geography and study settings. We fitted time series model to weekly national data available from the WHO influenza surveillance system (FluNet) to further characterize seasonal parameters.

Results

Influenza and RSV activity consistently peaked during winter months in temperate locales, while there was greater diversity in the tropics. Several temperate locations experienced semi-annual influenza activity with peaks occurring in winter and summer. Semi-annual activity was relatively common in tropical areas of Southeast Asia for both viruses. Biennial cycles of RSV activity were identified in Northern Europe. Both viruses exhibited weak latitudinal gradients in the timing of epidemics by hemisphere, with peak timing occurring later in the calendar year with increasing latitude (P<0.03). Time series model applied to influenza data from 85 countries confirmed the presence of latitudinal gradients in timing, duration, seasonal amplitude, and between-year variability of epidemics. Overall, 80% of tropical locations experienced distinct RSV seasons lasting 6 months or less, while the percentage was 50% for influenza.

Conclusion

Our review combining literature and electronic data sources suggests that a large fraction of tropical locations experience focused seasons of respiratory virus activity in individual years. Information on seasonal patterns remains limited in large undersampled regions, included Africa and Central America. Future studies should attempt to link the observed latitudinal gradients in seasonality of viral epidemics with climatic and population factors, and explore regional differences in disease transmission dynamics and attack rates.     

Priming with a secreted form of the fusion protein of respiratory syncytial virus (RSV) promotes interleukin-4 (IL-4) and IL-5 production but not pulmonary eosinophilia following RSV challenge

The attachment (G) protein of respiratory syncytial virus (RSV) is synthesized as two mature forms: a membrane-anchored form and a smaller secreted form. BALB/c mice scarified with vaccinia virus (VV) expressing the secreted form develop a greater pulmonary eosinophilic influx following RSV challenge than do mice scarified with VV expressing the membrane-anchored form. To determine if a soluble form of an RSV protein was sufficient to induce eosinophilia following RSV challenge, a cDNA that encoded a secreted form of the fusion (F) protein of RSV was constructed and expressed in VV (VV-Ftm(-)). Splenocytes and lung lymphocytes from mice primed with VV-Ftm(-) produced significantly more of the Th2 cytokines interleukin-4 (IL-4) and IL-5 than did mice vaccinated with VV expressing either the native (membrane-anchored) form of the F protein or the G protein. Although mice scarified with VV-Ftm(-) developed a slight increase in the number of pulmonary eosinophils following RSV infection, the increase was not as great as that seen in VV-G-primed mice. Despite the increased IL-4 and IL-5 production and in contrast to mice primed with VV-G, mice primed with VV-Ftm(-) developed RSV-specific cytotoxic T lymphocytes (CTL) and maintained high levels of gamma interferon production. These data demonstrate that recombinant VV strains expressing soluble forms of RSV proteins induce immune responses that are more Th2-like. However, this change alone does not appear sufficient to induce vaccine-augmented disease in the face of active CD8(+) CTL populations.     

Variable Immune-Driven Natural Selection in the Attachment (G) Glycoprotein of Respiratory Syncytial Virus (RSV)

A maximum-likelihood analysis of selection pressures acting on the attachment (G) glycoprotein gene of respiratory syncytial virus (RSV) from humans (HRSV) and bovines (BRSV) is presented. Six positively selected sites were identified in both group A and group B of HRSV, although only one site was common between them, while no positively selected sites were detected in BRSV. All positively selected sites were located within the ectodomain of the G protein and showed some association with positions of immunoglobulin (Ig) epitopes and sites of O-glycosylation. These results suggest that immune (antibody)-driven natural selection is an important determinant of RSV evolution and that this selection pressure differs among strains. The passage histories of RSV strains were also shown to affect the distribution of positively selected sites, particularly in HRSV B, and should be considered whenever retrospective analysis of adaptive evolution is undertaken. Received: 15 August 2000 / Accepted: 2 November 2000     

Effects of Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) in the Perinatal Lamb Model of RSV

Respiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in infants and children worldwide. There are currently no licensed vaccines or effective antivirals. The lack of a vaccine is partly due to increased caution following the aftermath of a failed clinical trial of a formalin-inactivated RSV vaccine (FI-RSV) conducted in the 1960’s that led to enhanced disease, necessitating hospitalization of 80% of vaccine recipients and resulting in two fatalities. Perinatal lamb lungs are similar in size, structure and physiology to those of human infants and are susceptible to human strains of RSV that induce similar lesions as those observed in infected human infants. We sought to determine if perinatal lambs immunized with FI-RSV would develop key features of vaccine-enhanced disease. This was tested in colostrum-deprived lambs immunized at 3–5 days of age with FI-RSV followed two weeks later by RSV infection. The FI-RSV-vaccinated lambs exhibited several key features of RSV vaccine-enhanced disease, including reduced RSV titers in bronchoalveolar lavage fluid and lung, and increased infiltration of peribronchiolar and perivascular lymphocytes compared to lambs either undergoing an acute RSV infection or naïve controls; all features of RSV vaccine-enhanced disease. These results represent a first step proof-of-principle demonstration that the lamb can develop altered responses to RSV following FI-RSV vaccination. The lamb model may be useful for future mechanistic studies as well as the assessment of RSV vaccines designed for infants.     

Lack of Interleukin-6 (IL-6) Enhances Susceptibility to Infection but Does Not Alter Latency or Reactivation of Herpes Simplex Virus Type 1 in IL-6 Knockout Mice

The ability of the pleotropic, proinflammatory cytokine interleukin-6 (IL-6) to affect the replication, latency, and reactivation of herpes simplex virus type 1 (HSV-1) in cell culture and in IL-6 knockout (KO) mice was studied. In initial studies, we found no effect of exogenous IL-6, monoclonal antibodies to IL-6, or monoclonal antibody to the IL-6 coreceptor, gp130, on HSV-1 replication in vitro by plaque assay or reactivation ex vivo by explant cocultivation of latently infected murine trigeminal ganglia (TG). Compared with the wild-type (WT) mice, the IL-6 KO mice were less able to survive an ocular challenge with 10(5) PFU of HSV-1 (McKrae) (40% survival of WT and 7% survival KO mice; P = 0.01). There was a sixfold higher 50% lethal dose of HSV-1 in WT than IL-6 KO mice (1.7 x 10(4) and 2.7 x 10(3) PFU, respectively). No differences were observed in titers of virus recovered from the eyes, TG, or brains or in the rates of virus reactivation by explant cocultivation of TG from latently infected WT or KO mice. Exposure of latently infected mice to UV light resulted in comparable rates of reactivation and in the proportions of WT and KO animals experiencing reactivation. Moreover, quantitative PCR assays showed nearly identical numbers of HSV-1 genomes in latently infected WT and IL-6 KO mice. These studies indicate that while IL-6 plays a role in the protection of mice from lethal HSV infection, it does not substantively influence HSV replication, spread to the nervous system, establishment of latency, or reactivation.     

Addition of a Missense Mutation Present in the L Gene of Respiratory Syncytial Virus (RSV) cpts530/1030 to RSV Vaccine Candidate cpts248/404 Increases Its Attenuation and Temperature Sensitivity

Respiratory syncytial virus (RSV) cpts530/1030 is an attenuated, temperature-sensitive subgroup A vaccine candidate derived previously from cold-passaged RSV (cpRSV) by two sequential rounds of chemical mutagenesis and biological selection. Here, cpts530/1030 was shown to be highly attenuated in the upper and lower respiratory tracts of seronegative chimpanzees. However, evaluation in seropositive children showed that it retains sufficient replicative capacity and virulence to preclude its direct use as a live attenuated vaccine. Nucleotide sequence analysis of the genome of cpts530/1030 showed that it had acquired two nucleotide substitutions (compared to its cpts530 parent), both of which were in the L gene: a silent mutation at nucleotide position 8821 (amino acid 108) and a missense mutation at nucleotide position 12458 resulting in a tyrosine-to-asparagine change at amino acid 1321, herein referred to as the 1030 mutation. It also contained the previously identified 530 missense mutation at nucleotide 10060 in the L gene. The genetic basis of attenuation of cpts530/1030 was defined by the introduction of the 530 and 1030 mutations into a cDNA clone of cpRSV, from which recombinant RSV was derived and analyzed to determine the contribution of each mutation to the temperature sensitivity (ts) and attenuation (att) phenotypes of cpts530/1030. The 530 mutation, derived from cpts530, was previously shown to be responsible for the ts and att phenotypes of that virus. In the present study, the 1030 mutation was shown to be responsible for the increased temperature sensitivity of cpts530/1030. In addition, the 1030 mutation was shown to be responsible for the increased level of attenuation of cpts530/1030 in the upper and lower respiratory tracts of mice. The 530 and 1030 mutations were additive in their effects on the ts and att phenotypes. It was possible to introduce the 1030 mutation, but not the 530 mutation, into an attenuated vaccine candidate with residual reactogenicity in very young infants, namely, cpts248/404, by use of reverse genetics. The inability to introduce the 530 mutation into the cpts248/404 virus was shown to be due to its incompatibility with the 248 missense mutation at the level of L protein function. The resulting rA2cp248/404/1030 mutant virus was more temperature sensitive and more attenuated than the cpts248/404 parent virus, making it a promising new RSV vaccine candidate created by use of reverse genetics to improve upon an existing vaccine virus.     

Parainfluenza virus type 3 expressing the native or soluble fusion (F) Protein of Respiratory Syncytial Virus (RSV) confers protection from RSV infection in African green monkeys

Respiratory syncytial virus (RSV) causes respiratory disease in young children, the elderly, and immunocompromised individuals, often resulting in hospitalization and/or death. After more than 40 years of research, a Food and Drug Administration-approved vaccine for RSV is still not available. In this study, a chimeric bovine/human (b/h) parainfluenza virus type 3 (PIV3) expressing the human PIV3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins from an otherwise bovine PIV3 (bPIV3) genome was employed as a vector for RSV antigen expression with the aim of generating novel RSV vaccines. b/h PIV3 vaccine candidates expressing native or soluble RSV F proteins were evaluated for efficacy and immunogenicity in a nonhuman primate model. b/h PIV3 is suited for development of pediatric vaccines since bPIV3 had already been evaluated in clinical studies in 1- and 2-month-old infants and was found to be safe, immunogenic, and nontransmissible in a day care setting (Karron et al., Pediatr. Infect. Dis. J. 15:650-654, 1996; Lee et al., J. Infect. Dis. 184:909-913, 2001). African green monkeys immunized with b/h PIV3 expressing either the native or soluble RSV F protein were protected from challenge with wild-type RSV and produced RSV neutralizing and RSV F-protein specific immunoglobulin G serum antibodies. The PIV3-vectored RSV vaccines evaluated here further underscore the utility of this vector system for developing safe and immunogenic pediatric respiratory virus vaccines.     

Mapping the Anatomy of Respiratory Syncytial Virus Infection of the Upper Airways in Chinchillas (Chinchilla lanigera)

Although most viral infections of the upper respiratory tract can predispose to bacterial otitis media, human respiratory syncytial virus (HRSV) is the predominant viral copathogen of this highly prevalent pediatric polymicrobial disease. Rigorous study of the specific mechanisms by which HRSV predisposes to otitis media has been hindered by lack of a relevant animal model. We recently reported that the chinchilla, the preferred rodent host for studying otitis media, is semipermissive for upper-airway HRSV infection. In the current study, we defined the anatomy and kinetics of HRSV infection and spread in the upper airway of chinchilla hosts. Chinchillas were challenged intranasally with a fluorescent-protein–expressing HRSV. Upper-airway tissues were recovered at multiple time points after viral challenge and examined by confocal microscopy and immunohistochemistry. HRSV replication was observed from the rostral- to caudalmost regions of the nasal cavity as well as throughout the Eustachian tube in a time-dependent manner. Although fluorescence was not observed and virus was not detected in nasopharyngeal lavage fluids 14 d after infection, the latest time point examined in this study, occasional clusters of immunopositive cells were present, suggesting that the nasal cavity may serve as a reservoir for HRSV. These data provide important new information concerning the time course of HRSV infection of the uppermost airway and suggest that chinchillas may be useful for modeling the HRSV-induced changes that predispose to secondary bacterial infection.Abbreviations: HRSV, human respiratory syncytial virus; rrHRSV, recombinant red fluorescent human respiratory syncytial virus; URT, upper respiratory tractHuman respiratory syncytial virus (HRSV), an enveloped, negative-strand, nonsegmented RNA virus of the family Paramyxoviridae, is the single greatest causative agent of acute respiratory tract infections in infants and children worldwide.²³ Although HRSV infection generally is limited to the upper respiratory tract (URT), in the United States, primary HRSV infection is associated with a 0.5% hospitalization rate for those children who develop severe bronchiolitis or pneumonia.⁹ One of the most interesting aspects of HRSV is its ubiquity: there are annual winter–spring outbreaks in temperate climates,⁵ and approximately 90% of all children have experienced infection by their second birthday.⁹ Although immunity to HRSV is sufficient to prevent reinfection of the lower airway in most human patients, this response is incomplete, resulting in reinfection of the upper airway throughout life.⁹ Although URT infection by HRSV alone does not constitute a serious problem for healthy adults, its association with the development of bacterial otitis media in children^{11-13,17-19,21,25,28,29,34} and exacerbation of asthma in all age groups¹⁶ make it an important health concern.Despite the ubiquity of the virus, the epidemiology of HRSV is not well understood. There is no known animal reservoir, and although new strains emerge over time, many remain in circulation over several seasons or reappear many years after they were first detected.^27,33 Therefore, although antigenic variation driven by development of HRSV immunity in a given population is possible, this hypothesis has not yet been proven. In fact, in one study,¹⁰ human subjects could be infected repeatedly with the same HRSV strain, and the presence of virus-specific antibody provided only short-lived and incomplete protection. Therefore, HRSV may circulate among seropositive persons, and it has been suggested that persistently infected persons may harbor the virus between seasonal outbreaks.^30,32 Therefore, in addition to the important clinical issues surrounding the prevention of HRSV disease, basic scientific questions regarding HRSV circulation and mechanisms of viral immunoevasion remain unanswered.A key hurdle in the study of HRSV pathogenesis has been the lack of a suitable animal model. Most published studies have used BALB/c mice, which have the advantage of many reagents available for the study of immune responses but the disadvantage of relative resistance to HRSV infection.²² Although pulmonary infection is easily detected in HRSV-infected mice, primary infection of the upper airway in this species is minimal^6,7 and secondary infection of the URT does not occur. More susceptible rodent species include the cotton rat (Sigmidon hispidus)²⁶ and chinchilla (Chinchilla lanigera),⁶ which are both relatively permissive for HRSV infection of the upper airway. Given the paucity of URT specimens encountered in general pathology practice, the development of a robust small animal model for the study of HRSV infection and spread in the uppermost airway is particularly important. Moreover, effective vaccine development depends on a better understanding of why this compartment remains susceptible to reinfection in immune hosts.Here we describe the anatomy of HRSV infection in the chinchilla URT over a 2-wk period, using confocal microscopy to monitor the retrograde spread of a recombinant red fluorescent protein-expressing HRSV construct (rrHRSV)⁸ from the site of inoculation. Although rrHRSV has previously been used to study the susceptibility of various cell types to virus infection in vitro,^8,36 our current report is the first wherein this biologic agent has been used to trace the route and extent of infection after intranasal instillation of virus in vivo. To establish the usefulness of our approach, immunohistochemistry and plaque assay were used to verify the sensitivity and specificity of fluorescence detected at 2, 3, 5, and 14 d after infection. By these combined methods, we were able to follow the retrograde spread of virus infection from the respiratory epithelium of the nasoturbinates and nasopharynx (at the earliest time point) to the Eustachian tubes and ethmoid turbinates at later time points. The ability to visualize the anatomy and kinetics of HRSV replication in the uppermost airway can now form the basis for future studies of upper-airway susceptibility to virus reinfection and bacterial coinfection.     

Interleukin-12 (IL-12) and IL-18 Are Important in Innate Defense against Genital Herpes Simplex Virus Type 2 Infection in Mice but Are Not Required for the Development of Acquired Gamma Interferon-Mediated Protective Immunity

Using a combination of gene-targeted mice and neutralizing antibodies, we showed that interleukin-12 (IL-12) and IL-18 are important in the innate control of genital herpes simplex virus type 2 infection but were not found to be critical, either singly or in combination, for the development of a protective gamma interferon-mediated immune response.     

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.     

Distinct Structural Requirements for Interleukin-4 (IL-4) and IL-13 Binding to the Shared IL-13 Receptor Facilitate Cellular Tuning of Cytokine Responsiveness

Both interleukin-4 (IL-4) and IL-13 can bind to the shared receptor composed of the IL-4 receptor α chain and the IL-13 receptor α1 chain (IL-13Rα1); however, the mechanisms by which these ligands bind to the receptor chains are different, enabling the principal functions of these ligands to be different. We have previously shown that the N-terminal Ig-like domain in IL-13Rα1, called the D1 domain, is the specific and critical binding unit for IL-13. However, it has still remained obscure which amino acid has specific binding capacity to IL-13 and why the D1 domain acts as the binding site for IL-13, but not IL-4. To address these questions, in this study we performed mutational analyses for the D1 domain, combining the structural data to identify the amino acids critical for binding to IL-13. Mutations of Lys-76, Lys-77, or Ile-78 in c′ strand in which the crystal structure showed interaction with IL-13, and those of Trp-65 and Ala-79 adjacent to the interacting site, resulted in significant impairment of IL-13 binding, demonstrating that these amino acids generate the binding site. Furthermore, mutations of Val-35, Leu-38, or Val-42 at the N-terminal β-strand also resulted in loss of IL-13 binding, probably from decreased structural stability. None of the mutations employed here affected IL-4 binding. These results demonstrate that the D1 domain of IL-13Rα1 acts as an affinity converter, through direct cytokine interactions, that allows the shared receptor to respond differentially to IL-4 and IL-13.Interleukin-4 (IL-4)² and IL-13 are related cytokines. IL-4 binds to a heterodimeric complex composed of the IL-4R α chain (IL-4Rα) and the common γ chain (γc), or of IL-4Rα and the IL-13R α1 chain (IL-13Rα1), called type I or type II IL-4R, respectively (1, 2). In contrast, IL-13 binds to type II IL-4R, but not type I IL-4R. Therefore, type II IL-4R is also called IL-13R. This means that IL-4 and IL-13 share the same receptor, type II IL-4R·IL-13R, which explains why IL-4 and IL-13 exert similar activities. However, the principal functions of IL-4 and IL-13 are different. As type I IL-4R is mainly expressed on hematopoietic cells, IL-4 acts on these cells, inducing Th2 differentiation in T cells and immunoglobulin class switching to IgE in B cells (1, 3). In contrast, type II IL-4R·IL-13R expresses ubiquitously, including nonhematopoietic cells, and IL-13 plays a central role in the pathogenesis of bronchial asthma by acting on these cells, including epithelial cells and fibroblasts (1, 4). Thus, it can be said that the principal role of IL-4 is an immunoregulatory cytokine, whereas that of IL-13 is an effector cytokine (5).The assembly mechanism for the binding of either IL-4 or IL-13 to type II IL-4R·IL-13R is unique. IL-4 first binds to IL-4Rα with high affinity (K_d = 1 nm), followed by recruitment of IL-13Rα1 with low affinity. In contrast, IL-13 first binds to IL-13Rα1 with low affinity (K_d = 30–37 nm), and then the complex recruits IL-4Rα, forming a high affinity receptor (K_d = 0.03–0.4 nm (6, 7)). This means that, although both IL-4 and IL-13 use IL-4Rα and IL-13Rα1, the roles of IL-4Rα and IL-13Rα1 as the primary or secondary binding unit are the opposite of those for IL-4 and IL-13. Furthermore, these differences in affinity between the ligand, the primary binding unit, and the secondary binding unit can result in that in nonhematopoietic cells on which IL-131 is expressed more abundantly than IL-4α, the number of the IL-13 receptor complex continues to rise as the IL-13 concentration increases, whereas the formation of the IL-4 receptor complex is saturated at a low IL-4 concentration. This can explain why IL-13 transduces stronger signals than IL-4 in nonhematopoietic cell such as epithelial cells and fibroblasts.We previously found that the N-terminal Ig-like domain in IL-13Rα1, called the D1 domain, is the specific and critical binding unit for IL-13, but not for IL-4, using the D1 domain-deleted IL-13Rα1 (8). LaPorte et al. recently described the crystal structure of the IL-13·IL-13Rα1·IL-4Rα, showing that the c′ strand of the D1 domain of IL-13Rα1 and the C-D strand of IL-13 generate an antiparallel β-sheet structure (7). Furthermore, this structural analysis showed that the polar bonds between IL-4 and IL-4Rα were diminished in the IL-13·IL-4Rα complex, possibly suggesting why IL-4Rα has high and no affinity with IL-4 and IL-13, respectively. These results confirmed that the unique assembly mechanism of type II IL-4R·IL-13R for IL-4 and IL-13 is dictated by the D1 domain and indicated that the c′ strand in the D1 domain is the binding site of IL-13Rα1 to IL-13. It is thought that IL-13Rα1 has evolved from γc, which does not have the extra Ig domain, acquiring the D1 domain probably from IL-2Rα or IL-15Rα (7, 9). In other words, the acquisition of the D1 domain enables the cells to respond to IL-13 in addition to IL-4. In this sense, the D1 domain appears to be an affinity converter that has evolved differential interactions with IL-4 and IL-13 to respond to the two cytokines distinctly, based on receptor expression levels and cytokine concentration. Thus, the evolution of distinct interactions of D1 with IL-4 versus IL-13 is an unprecedented example of divergent evolution of function of the same structure. Interestingly, in the structural study, it was observed that the c′ strand of the D1 domain of IL-13Rα1 can also generate an antiparallel β-sheet structure with IL-4 that appears similar to that of IL-13 (7), leaving open the question of whether it is energetically important for IL-13 but not IL-4, and whether direct interactions are required.From these studies, several questions remain unresolved. The structures did not make it clear if this differential effect is indirect, or through direct interaction with the cytokines. Are the c′ contacts with cytokines energetically important and distinct for IL-4 and IL-13? If this is the case, then the second question is which amino acid in the c′ strand has specific binding capacity to IL-13. The third question is why does this portion act as the binding site specific for IL-13, but not IL-4. To address these questions, we took advantage of the mutational approach for the D1 domain, combining data from the structural study, and identified the amino acids critical for binding to IL-13.