Chimeric bovine respiratory syncytial virus with glycoprotein gene substitutions from human respiratory syncytial virus (HRSV): effects on host range and evaluation as a live-attenuated HRSV vaccine

We recently developed a system for the generation of infectious bovine respiratory syncytial virus (BRSV) from cDNA. Here, we report the recovery of fully viable chimeric recombinant BRSVs (rBRSVs) that carry human respiratory syncytial virus (HRSV) glycoproteins in place of their BRSV counterparts, thus combining the replication machinery of BRSV with the major antigenic determinants of HRSV. A cDNA encoding the BRSV antigenome was modified so that the complete G and F genes, including the gene start and gene end signals, were replaced by their HRSV A2 counterparts. Alternatively, the BRSV F gene alone was replaced by that of HRSV Long. Each antigenomic cDNA directed the successful recovery of recombinant virus, yielding rBRSV/A2 and rBRSV/LongF, respectively. The HRSV G and F proteins or the HRSV F in combination with BRSV G were expressed efficiently in cells infected with the appropriate chimeric virus and were efficiently incorporated into recombinant virions. Whereas BRSV and HRSV grew more efficiently in bovine and human cells, respectively, the chimeric rBRSV/A2 exhibited intermediate growth characteristics in a human cell line and grew better than either parent in a bovine line. The cytopathology induced by the chimera more closely resembled that of BRSV. BRSV was confirmed to be highly restricted for replication in the respiratory tract of chimpanzees, a host that is highly permissive for HRSV. Interestingly, the rBRSV/A2 chimeric virus was somewhat more competent than BRSV for replication in chimpanzees but remained highly restricted compared to HRSV. This showed that the substitution of the G and F glycoproteins alone was not sufficient to induce efficient replication in chimpanzees. Thus, the F and G proteins contribute to the host range restriction of BRSV but are not the major determinants of this phenotype. Although rBRSV/A2 expresses the major neutralization and protective antigens of HRSV, chimpanzees infected with this chimeric virus were not significantly protected against subsequent challenge with wild-type HRSV. This suggests that the growth restriction of rBRSV/A2 was too great to provide adequate antigen expression and that the capacity of this chimeric vaccine candidate for replication in primates will need to be increased by the importation of additional HRSV genes.     

The envelope proteins from purified respiratory syncytial virus protect mice from intranasal virus challenge

A lyophilized subunit vaccine prepared from purified respiratory syncytial virus, which contained the envelope glycoproteins F and G and the nonglycosylated matrix protein VPM, was tested in SJL mice for its ability to protect the lungs of mice from intranasal viral challenge. Initially, the mice were injected subcutaneously with one, two, or three doses of 5 or 25 micrograms of vaccine in 50% complete Freund's adjuvant or with complete Freund's adjuvant or phosphate-buffered saline only. Although none of the mice produced neutralizing serum antibody, three doses of 25 micrograms elicited antibodies to F, G, and VPM. Despite the absence of detectable neutralizing antibodies, the lungs of 93% of the vaccinated mice were protected from intranasal viral challenge. Because the initial protocol did not elicit neutralizing antibodies and a few single-dose animals were not protected, a second vaccine trial was carried out. For these studies the priming dose was increased to 50 micrograms, which was followed, in half the vaccine recipients, by a second dose of 25 micrograms. Mice given the priming dose of vaccine produced antibody to G and showed no neutralizing activity, whereas the mice given two doses of vaccine produced antibodies to G, F, and VPM and also displayed neutralizing activity for respiratory syncytial virus. The lungs of 100% of the vaccine recipients in this trial were protected from intranasal challenge. Although the vaccine elicited antibody to VPM, this response did not correlate with protection. In addition, examination of the sera from unimmunized mice recovering from respiratory syncytial virus infection revealed a serum antibody profile similar to that noted for humans, lacking antibody to VPM. Thus, the data show that a combined glycoprotein subunit vaccine affords complete protection to viral challenge and offers an approach to develop a multivalent subunit vaccine.     

Immunoprophylaxis of respiratory syncytial virus infection in the infant ferret.

Infant ferrets can be protected from respiratory syncytical virus challenge at 3 days of age by gestational infection of their mothers. Ferrets acquire their immunity to respiratory syncytial virus postpartum via immunizing products of lactation. The level of protection against viral replication correlates with the maternal serum neutralizing titer or a concomitant factor. Passive administration of adult ferret serum with a neutralizing titer of 1:1024 or greater, either i.p. or orally does not confer immunity. A nonantibody-mediated protective mechanism appears to play an important role in protecting the infant ferret from respiratory syncytial virus replication. Our findings allow the testing of the efficacy of future human vaccines before human clinical trial.     

Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response

The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-alpha) receptor. Treatment of cells with recombinant universal IFN-alpha A/D or IFN-beta revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-alpha/beta mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.     

Current progress on development of respiratory syncytial virus vaccine

Human respiratory syncytial virus (HRSV) is a major cause of upper and lower respiratory tract illness in infants and young children worldwide. Despite its importance as a respiratory pathogen, there is currently no licensed vaccine for prophylaxis of HRSV infection. There are several hurdles complicating the development of a RSV vaccine: 1) incomplete immunity to natural RSV infection leading to frequent re-infection, 2) immature immune system and maternal antibodies of newborn infants who are the primary subject population, and 3) imbalanced Th2-biased immune responses to certain vaccine candidates leading to exacerbated pulmonary disease. After the failure of an initial trial featuring formalin-inactivated virus as a RSV vaccine, more careful and deliberate efforts have been made towards the development of safe and effective RSV vaccines without vaccine-enhanced disease. A wide array of RSV vaccine strategies is being developed, including live-attenuated viruses, protein subunit-based, and vector-based candidates. Though licensed vaccines remain to be developed, our great efforts will lead us to reach the goal of attaining safe and effective RSV vaccines in the near future.     

Human respiratory syncytial virus glycoprotein G expressed from a recombinant vaccinia virus vector protects mice against live-virus challenge.

Recombinant vaccinia virus vectors were constructed which expressed the major surface glycoprotein G of human respiratory syncytial (RS) virus. The biological activity of the G protein expressed from these vectors was assayed. Inoculation of rabbits with live recombinant virus induced high titers of antibody which specifically immunoprecipitated RS virus G protein and was capable of neutralizing RS virus infectivity. Immunization of mice by either the intranasal or the intraperitoneal route with recombinant virus that expressed only the G protein resulted in complete protection of the lower respiratory tract upon subsequent challenge with live RS virus.     

Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells

Bovine respiratory syncytial virus (BRSV) escapes from cellular responses to alpha/beta interferon (IFN-alpha/beta) by a concerted action of the two viral nonstructural proteins, NS1 and NS2. Here we show that the NS proteins of human RSV (HRSV) are also able to counteract IFN responses and that they have the capacity to protect replication of an unrelated rhabdovirus. Even combinations of BRSV and HRSV NS proteins showed a protective activity, suggesting common mechanisms and cellular targets of HRSV and BRSV NS proteins. Although able to cooperate, NS proteins from BRSV and HRSV showed differential protection capacity in cells from different hosts. A chimeric BRSV with HRSV NS genes (BRSV h1/2) was severely attenuated in bovine IFN competent MDBK and Klu cells, whereas it replicated like BRSV in IFN-incompetent Vero cells or in IFN-competent human HEp-2 cells. After challenge with exogenous IFN-alpha, BRSV h1/2 was better protected than wild-type BRSV in human HEp-2 cells. In contrast, in cells of bovine origin, BRSV h1/2 was much less resistant to exogenous IFN than wild-type BRSV. These data demonstrate that RSV NS1 and NS2 proteins are major determinants of host range. The differential IFN escape capacity of RSV NS proteins in cells from different hosts provides a basis for rational development of attenuated live RSV vaccines.     

Modulation of protective immunity, eosinophilia, and cytokine responses by selective mutagenesis of a recombinant G protein vaccine against respiratory syncytial virus

Using an Escherichia coli-grown plasmid vector encoding a fragment of thioredoxin (Trx) fused to a central region (amino acids 128 to 229) of the respiratory syncytial virus (RSV) (Long strain) G protein, we employed site-directed mutagenesis to investigate the importance of selected amino acids to vaccine efficacy. Mice were immunized with a total of 10 wild-type or mutant Trx-G proteins and challenged intranasally with RSV. Striking differences in the induction of RSV G-protein-specific antibodies, protection against RSV challenge, cytokine RNA responses, and induction of RSV-associated eosinophilic inflammation were observed among the mutant proteins examined. Taken together, the results identify a critical role for specific amino acids in the induction of protective immunity and priming for eosinophilia against RSV.     

Role of alpha/beta interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking NS proteins

Alpha/beta interferons (IFN-alpha/beta) are not only a powerful first line of defense against pathogens but also have potent immunomodulatory activities. Many viruses have developed mechanisms of subverting the IFN system to enhance their virulence. Previous studies have demonstrated that the nonstructural (NS) genes of bovine respiratory syncytial virus (BRSV) counteract the antiviral effects of IFN-alpha/beta. Here we demonstrate that, in contrast to wild-type BRSVs, recombinant BRSVs (rBRSVs) lacking the NS proteins, and those lacking NS2 in particular, are strong inducers of IFN-alpha/beta in bovine nasal fibroblasts and bronchoalveolar macrophages. Furthermore, whereas the NS deletion mutants replicated to wild-type rBRSV levels in cells lacking a functional IFN-alpha/beta system, their replication was severely attenuated in IFN-competent cells and in young calves. These results suggest that the NS proteins block the induction of IFN-alpha/beta gene expression and thereby increase the virulence of BRSV. Despite their poor replication in the respiratory tract of young calves, prior infection with virus lacking either the NS1 or the NS2 protein induced serum antibodies and protection against challenge with virulent BRSV. The greater level of protection induced by the NS2, than by the NS1, deletion mutant, was associated with higher BRSV-specific antibody titers and greater priming of BRSV-specific, IFN-gamma-producing CD4(+) T cells. Since there were no detectable differences in the ability of these mutants to replicate in the bovine respiratory tract, the greater immunogenicity of the NS2 deletion mutant may be associated with the greater ability of this virus to induce IFN-alpha/beta.     

Vaccine-induced immunopathology during bovine respiratory syncytial virus infection: exploring the parameters of pathogenesis

The bovine and human respiratory syncytial viruses cause severe lower respiratory tract infections. Effective vaccines against the respiratory syncytial viruses have been lacking since vaccine failures in the 1960s and 1970s. In this report, we describe a bovine respiratory syncytial virus (bRSV) challenge model in which both classical bRSV respiratory infection and vaccine-enhanced immune pathology were reproduced. The classical, formalin-inactivated (FI) bRSV vaccine that has been associated with vaccine failure was efficient in inducing high antibody titers and reducing viral loads but also primed calves for a far more serious enhanced respiratory disease after a bRSV challenge, thereby mimicking the enhanced clinical situation in FI human RSV (hRSV)-immunized and hRSV-infected infants in the 1960s. We show that immunization with FI-bRSV mainly primes a Th2-like inflammatory response that is characterized by a significant eosinophilic influx in the bronchial alveolar lung fluid and lung tissues and high levels of immunoglobulin E serum antibodies. The current model may be useful in the evaluation of new bRSV candidate vaccines for potency and safety.     

Evaluation of protective efficacy of respiratory syncytial virus vaccine against A and B subgroup human isolates in Korea

Human respiratory syncytial virus (HRSV) is a significant cause of upper and lower respiratory tract illness mainly in infants and young children worldwide. HRSV is divided into two subgroups, HRSV-A and HRSV-B, based on sequence variation within the G gene. Despite its importance as a respiratory pathogen, there is currently no safe and effective vaccine for HRSV. In this study, we have detected and identified the HRSV by RT-PCR from nasopharyngeal aspirates of Korean pediatric patients. Interestingly, all HRSV-B isolates exhibited unique deletion of 6 nucleotides and duplication of 60 nucleotides in the G gene. We successfully amplified two isolates ('KR/A/09-8' belonging to HRSV-A and 'KR/B/10-12' to HRSV-B) on large-scale, and evaluated the cross-protective efficacy of our recombinant adenovirus-based HRSV vaccine candidate, rAd/3xG, by challenging the immunized mice with these isolates. The single intranasal immunization with rAd/3xG protected the mice completely from KR/A/09-8 infection and partially from KR/B/10-12 infection. Our study contributes to the understanding of the genetic characteristics and distribution of subgroups in the seasonal HRSV epidemics in Korea and, for the first time, to the evaluation of the cross-protective efficacy of RSV vaccine against HRSV-A and -B field-isolates.     

Biophysical studies of respiratory syncytial virus. I. Density of respiratory syncytial virus and associated complement-fixing antigens in cesium chloride density gradient

Coates, Helen V. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), Ben R. Forsyth, and R. M. Chanock. Biophysical studies of respiratory syncytial virus. I. Density of respiratory syncytial virus and associated complement-fixing antigens in a cesium chloride density gradient. J. Bacteriol. 91:1263-1269. 1966.-Concentrated fluids from respiratory syncytial (RS) virus-infected tissue cultures (HEp-2 and BEK) were subjected to equilibrium sedimentation in cesium chloride. When two antigenically distinct strains of RS virus (Long and 18537) were tested, approximately 90% of the infectious virus was recovered in a sharp, symmetrical peak with a density of 1.22 to 1.24. In a similar study, unconcentrated virus had a density of 1.25 to 1.27. Two immunologically distinguishable complement-fixing antigens (antigens A and B) were detected at densities of 1.28 to 1.32 and 1.23 to 1.37. In addition, the existence of a third antigen (density of 1.22 to 1.30) was suggested. The possible origin of these antigens is discussed relative to the known properties of RS virus and the other myxoviruses.     

Response of vaccinated and unvaccinated bighorn sheep (Ovis canadensis canadensis) to experimental respiratory syncytial virus challenge

Five Rocky Mountain bighorn sheep (Ovis canadensis canadensis), approximately 5 mo old and without detectable antibody titers to respiratory syncytial virus (RSV), were assigned to two groups to study the effects of RSV challenge inoculation in vaccinated (n = 3) and unvaccinated (n = 2) bighorns. The three lambs vaccinated with a modified live bovine RSV vaccine developed a detectable antibody response to the vaccine. Vaccinated and unvaccinated lambs challenged with an ovine isolate of RSV developed increased levels of neutralizing antibody, but clinical signs of disease were not observed. Neutralizing antibody titers to RSV remained higher (2-4-fold) in vaccinated lambs over time when compared to unvaccinated lambs.     

Evolution of bovine respiratory syncytial virus

Until now, the analysis of the genetic diversity of bovine respiratory syncytial virus (BRSV) has been based on small numbers of field isolates. In this report, we determined the nucleotide and deduced amino acid sequences of regions of the nucleoprotein (N protein), fusion protein (F protein), and glycoprotein (G protein) of 54 European and North American isolates and compared them with the sequences of 33 isolates of BRSV obtained from the databases, together with those of 2 human respiratory syncytial viruses and 1 ovine respiratory syncytial virus. A clustering of BRSV sequences according to geographical origin was observed. We also set out to show that a continuous evolution of the sequences of the N, G, and F proteins of BRSV has been occurring in isolates since 1967 in countries where vaccination was widely used. The exertion of a strong positive selective pressure on the mucin-like region of the G protein and on particular sites of the N and F proteins is also demonstrated. Furthermore, mutations which are located in the conserved central hydrophobic part of the ectodomain of the G protein and which result in the loss of four Cys residues and in the suppression of two disulfide bridges and an alpha helix critical to the three-dimensional structure of the G protein have been detected in some recent French BRSV isolates. This conserved central region, which is immunodominant in BRSV G protein, thus has been modified in recent isolates. This work demonstrates that the evolution of BRSV should be taken into account in the rational development of future vaccines.     

Vaccination with recombinant alphavirus or immune-stimulating complex antigen against respiratory syncytial virus

Respiratory syncytial virus (RSV) causes severe respiratory diseases in infants and young children. Inappropriate immunity to the virus can lead to disease enhancement upon subsequent infection. In this study, we have characterized the antiviral immunity elicited by the recombinant Semliki Forest virus (SFV) encoding the RSV fusion (F) and attachment (G) protein, and compared with that induced by the immune-stimulating complex (ISCOM)-incorporated FG proteins. Antiviral immunity against RSV elicited nasally or parentally by either of the immunogen having divergent profiles could reduce lung RSV titers upon challenge. However, resistance to RSV without disease enhancement was only observed in those vaccinated with SFV recombinants via nasal route. Presence of postvaccination pulmonary IFN-gamma response to the H-2K(d)-restricted T cell epitope (F(85-93); KYKNAVTEL) was found to be associated with absence of enhanced pulmonary disease and goblet cell hyperplasia as well as reduced Th2-cytokine expression. This result demonstrates that the SFV recombinants can result in enhanced clearance of RSV without enhancing the RSV-associated disease, and underlines the importance in priming pulmonary MHC class I-restricted T cells when RSV FG-based vaccines are used.     

Circulating strains of human respiratory syncytial virus in central and south America

Human respiratory syncytial virus (HRSV) is a major cause of viral lower respiratory tract infections among infants and young children. HRSV strains vary genetically and antigenically and have been classified into two broad subgroups, A and B (HRSV-A and HRSV-B, respectively). To date, little is known about the circulating strains of HRSV in Latin America. We have evaluated the genetic diversity of 96 HRSV strains by sequencing a variable region of the G protein gene of isolates collected from 2007 to 2009 in Central and South America. Our results show the presence of the two antigenic subgroups of HRSV during this period with the majority belonging to the genotype HRSV-A2.     

Respiratory syncytial virus (RSV) fusion protein subunit F2, not attachment protein G,determines the specificity of RSV infection

Human respiratory syncytial virus (HRSV) and bovine RSV (BRSV) infect human beings and cattle in a species-specific manner. We have here analyzed the contribution of RSV envelope proteins to species-specific entry into cells. In contrast to permanent cell lines, primary cells of human or bovine origin, including differentiated respiratory epithelia, peripheral blood lymphocytes, and macrophages, showed a pronounced species-specific permissiveness for HRSV and BRSV infection, respectively. Recombinant BRSV deletion mutants lacking either the small hydrophobic (SH) protein gene or both SH and the attachment glycoprotein (G) gene retained their specificity for bovine cells, whereas corresponding mutants carrying the HRSV F gene specifically infected human cells. To further narrow the responsible region of F, two reciprocal chimeric F constructs were assembled from BRSV and HRSV F1 and F2 subunits. The specificity of recombinant RSV carrying only the chimeric F proteins strictly correlated with the origin of the membrane-distal F2 domain. A contribution of G to the specificity of entry could be excluded after reintroduction of BRSV or HRSV G. Virus with F1 and G from BRSV and with only F2 from HRSV specifically infected human cells, whereas virus expressing F1 and G from HRSV and F2 from BRSV specifically infected bovine cells. The introduction of G enhanced the infectiousness of both chimeric viruses to equal degrees. Thus, the role of the nominal attachment protein G is confined to facilitating infection in a non-species-specific manner, most probably by binding to cell surface glycosaminoglycans. The identification of the F2 subunit as the determinant of RSV host cell specificity facilitates identification of virus receptors and should allow for development of reagents specifically interfering with RSV entry.     

Quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats

The amount of passively acquired serum respiratory syncytial virus (RSV)-neutralizing antibodies required to protect the respiratory tract of cotton rats against infection was studied. Infant cotton rats were inoculated intraperitoneally with various dilutions of a single pool of sera derived from cotton rats convalescent from RSV infection. After 24 h, these animals were inoculated with RSV intranasally. Virus replication in the respiratory tract was suppressed in cotton rats which had a serum neutralizing antibody titer of 1:100 or greater. Resistance was greater in the lungs than in the nose. Complete or almost complete resistance in the lungs was observed in cotton rats with a serum neutralizing antibody titer of 1:380 or greater. The level of serum RSV-neutralizing antibodies required to confer significant resistance to infection in the cotton rat was similar to the level of maternally derived serum antibodies possessed by human infants less than 2 months of age, who as a group exhibit relative resistance to RSV disease compared with infants 2 to 6 months of age.     

Formulation with CpG oligodeoxynucleotides prevents induction of pulmonary immunopathology following priming with formalin-inactivated or commercial killed bovine respiratory syncytial virus vaccine

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-gamma production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.     

Neutralization of respiratory syncytial virus after cell attachment.

Little is known about the mechanisms of antibody-mediated neutralization of respiratory syncytial virus (RSV) which causes recurrent infections in human despite the virtually universal presence of neutralizing serum antibodies. Human serum neutralization titers showed strong correlation with post-cell-attachment neutralizing titers for both RSV-convalescent sera and control sera but showed less strong correlation with cell-attachment blocking titers. Neutralization was effective for the first 60 min of infection, indicating that immune serum-mediated neutralization of RSV infection largely involves inhibition of early events following cell attachment.