Although respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naive infants.However, designing an LAV with appropriate attenuation yet sufficient immunogenicity has proven challenging. In this study, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for nonstructural proteins NS1 and NS2 (dNS), deletion of the small hydrophobic protein (⌬SH) gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate, RSV-A2-dNS-⌬SH-BAF (DB1), was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log 10 total PFU and 2.7 log 10 PFU/g of tissue, respectively, compared to those in unvaccinated animals (P < 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity. IMPORTANCERSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naive infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. In this study, we introduced a novel strategy to attenuate a recombinant RSV vaccine by incorporating a low-fusion, subgroup B F protein in the genetic background of codondeoptimized nonstructural protein genes and a deleted small hydrophobic protein gene. The resultant vaccine candidate, DB1, was attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. R espiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants (1). Globally, RSV causes an estimated 3.4 million (1) hospitalizations and 234,000 deaths per year in children under the age of 5 years (2). Almost all children have been infected with RSV by the age of 2 years, with clinical manifestations ranging from upper respiratory tract infections to pneumonia with respiratory failure. Despite the striking burden of RSV disease in children worldwide, no RSV-specific therapies or vaccines are commercially available. The development of a safe and effective RSV vaccine is therefore a public health priority.The initial attempt to develop an RSV vaccine by formalin inactivation (FI-RSV) not only failed to protect ...

The consistent presence of the Epstein-Barr virus (EBV) genome in certain malignancies, particularly its nearly universal presence in AIDS-related central nervous system lymphomas (4, 6, 29) and nasopharyngeal carcinomas (31), suggests that EBV itself could serve as a target for the preferential killing of tumor cells using gene delivery methods. Although the use of adenovirus vectors expressing EBV-specific toxins could potentially be useful for treating EBV-positive epithelial cell tumors, EBV-associated B-cell tumors are unlikely to be susceptible to conventional adenovirus-mediated delivery. The recently identified adenovirus receptor for serogroups 2 and 5, the coxsackievirus-adenovirus receptor (CAR), is not expressed in most hematologic cell lines (19,28,44), and consequently, adenovirus delivery to most B-cell lines is extremely inefficient. Nevertheless, other advantageous aspects of recombinant adenovirus vectors (rAd), including the extremely high achievable titers and the large size (10 kb) of the gene inserts tolerated, continue to make rAd the most attractive currently available gene delivery vectors. Therefore, there has been intense interest in modifying rAd to improve their delivery into hematopoietic cell types.Bispecific antibodies (BsAb) are covalently linked antibodies with distinct specificities (40). BsAb can extend a virus's normal tropism by using specific antibodies to the virus's receptor (the fiber protein, in the case of adenovirus [41]) and an alternate cellular ligand. For example, the delivery of rAd with a FLAG epitope-modified adenovirus fiber protein to T cells was shown to be greatly enhanced when a bispecific antibody directed against the FLAG epitope and the T-cell-specific CD3 cell surface receptor (48) was used. The method has been applied to other virus-cell surface ligand systems (5,7,49).In this study, we have investigated the use of an anti-CD70-antifiber BsAb to enhance adenovirus delivery to CD70-positive B-cell lines. CD70 expression is usually limited to a small subset of highly activated B and T cells (42). In contrast, EBV-immortalized B cells (lymphoblastoid cell lines [LCLs]) routinely express CD70 (42), as do a number of EBV-positive, as well as EBV-negative, B-cell lymphomas (17,27). Expression of CD70 (which has been identified as the CD27 ligand) on T cells appears to have a physiological role in inducing CD27 ϩ B cells to proliferate and differentiate into plasma cells (2), while on B cells, CD70 seems to have a costimulatory effect upon T cells (3). Interestingly, although CD70 expression in vivo is usually limited to a few highly activated B cells and T cells, CD70 is also expressed in EBV-positive nasopharyngeal carcinomas (1, 31). Thus, CD70 expression could potentially

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