System Using Tandem Repeats of the cA Peptidoglycan-Binding Domain from Lactococcus lactis for Display of both N- and C-Terminal Fusions on Cell Surfaces of Lactic Acid Bacteria

Here, we established a system for displaying heterologous protein to the C terminus of the peptidoglycan-binding domain (cA domain) of AcmA (a major autolysin from Lactococcus lactis). Western blot and flow cytometric analyses revealed that the fusion proteins (cA-AmyA) of the cA domain and α-amylase from Streptococcus bovis 148 (AmyA) are efficiently expressed and successfully displayed on the surfaces of L. lactis cells. AmyA was also displayed on the cell surface while retaining its activity. Moreover, with an increase in the number of cA domains, the quantity of cA-AmyA fusion proteins displayed on the cell surface increased. When three repeats of the cA domain were used as an anchor protein, 82% of α-amylase activity was detected on the cells. The raw starch-degrading activity of AmyA was significantly higher when AmyA was fused to the C terminus of the cA domain than when it was fused to the N terminus. In addition, cA-AmyA fusion proteins were successfully displayed on the cell surfaces of Lactobacillus plantarum and Lactobacillus casei.     

Display of heterologous proteins on the surface of Lactococcus lactis using the H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix

Aims: The aim of this study was to develop a cell‐surface display system for foreign antigens on the surface of a Lactococcus lactis strain using an H and W domain of PrtB from Lactobacillus delburueckii subsp. bulgaricus as an anchoring matrix. Methods and Results: To construct a cell‐surface display pACL1 vector, a derivative of pSECE1 vector, we amplified the H and W domain of the cell‐surface proteinase Prt B from Lact. bulgaricus using specific primers and then cloned it into a site downstream of the secretion signal sequence in the pSECE1 vector. The new system, designed for cell‐surface display of recombinant proteins on L. lactis, was evaluated by the expression and display of the FliC protein of Salmonella enterica serovar Enteritidis as a reporter gene (pALC1:FliC). The expression of the FliC protein by the transformed cells was analysed by Western blot analysis, and the localization of FliC on the cell surface was confirmed by immunofluorescence microscopy and flow cytometry analysis. A specific band corresponding in size (approx. 110 kDa) to FliC plus the anchor residues was detected by anti‐FliC antibody in the cell extract of L. lactis H61 harbouring pALC1:FliC, but not L. lactis H61 harbouring pALC1. In addition, flow cytometry and immunofluorescence microscopy revealed FliC‐specific positive signals and a significant increase of fluorescence, respectively, in cells harbouring pALC1:FliC compared with that in control cells harbouring the parental pALC1 plasmid. These findings demonstrated that FliC was successfully displayed on the cell surface by the anchor domain of PrtB. Conclusions: A pALC1 vector using the H and W domain of PrtB from Lact. bulgaricus as an anchoring matrix can be used to successfully display the FliC protein on the surface of L. lactis. Significance and Impact of the Study: This novel way of displaying heterologous proteins on the cell surface of L. lactis using the PrtB anchor domain should prove useful for the delivery of antigens and other proteins.     

Characterization of potent RSV neutralizing antibodies isolated from human memory B cells and identification of diverse RSV/hMPV cross-neutralizing epitopes

ABSTRACT

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children and older adults. Currently, no licensed vaccine is available, and therapeutic options are limited. The primary target of neutralizing antibodies to RSV is the surface fusion (F) glycoprotein. Understanding the recognition of antibodies with high neutralization potencies to RSV F antigen will provide critical insights in developing efficacious RSV antibodies and vaccines. In this study, we isolated and characterized a panel of monoclonal antibodies (mAbs) with high binding affinity to RSV prefusion F trimer and neutralization potency to RSV viruses. The mAbs were mapped to previously defined antigenic sites, and some that mapped to the same antigenic sites showed remarkable diversity in specificity, binding, and neutralization potencies. We found that the isolated site III mAbs shared highly conserved germline V-gene usage, but had different cross-reactivities to human metapneumovirus (hMPV), possibly due to the distinct modes/angles of interaction with RSV and hMPV F proteins. Furthermore, we identified a subset of potent RSV/hMPV cross-neutralizing mAbs that target antigenic site IV and the recently defined antigenic site V, while the majority of the mAbs targeting these two sites only neutralize RSV. Additionally, the isolated mAbs targeting site Ø were mono-specific for RSV and showed a wide range of neutralizing potencies on different RSV subtypes. Our data exemplify the diversity of anti-RSV mAbs and provide new insights into the immune recognition of respiratory viruses in the Pneumoviridae family.     

Immunodominant T-cell epitope on the F protein of respiratory syncytial virus recognized by human lymphocytes.

The lymphocyte proliferative responses to respiratory syncytial virus (RSV) were evaluated for 10 healthy adult donors and compared with proliferative responses to a chimeric glycoprotein (FG glycoprotein) which consists of the extracellular domains of both the F and G proteins of RSV and which is produced from a recombinant baculovirus. The lymphocytes of all 10 donors responded to RSV, and the proliferative responses to the whole virus were highly correlated with the responses to the FG glycoprotein. These data suggested that one or both of these glycoproteins of RSV were major target structures for stimulation of the human lymphocyte proliferative response among virus-specific memory T cells. The lymphocytes of four donors were evaluated further for their proliferative responses to a nested set of overlapping peptides modeled on the extracellular and cytoplasmic domains of the F protein of RSV. Strikingly, the lymphocytes of all 4 donors responded primarily to a region defined by a single peptide spanning residues 338 to 355, and the lymphocytes of 2 donors responded to an overlapping peptide spanning residues 328 to 342 also, thus defining a region of the F1 subunit within residues 328 to 355 that may circumscribe an immunodominant site for stimulation of human T cells from a variety of individuals. This region of the F protein is highly conserved among A and B subgroup viruses. As revealed by monoclonal antibody blocking studies, the lymphocytes responding to this antigenic site had characteristics consistent with T helper cells. Similar epitope mapping studies were performed with BALB/c mice immunized with the FG protein in which a relatively hydrophobic peptide spanning residues 51 to 65 within the F2 subunit appeared to be the major T cell recognition determinant. The data are discussed with respect to an antigenic map of the F protein and the potential construction of a synthetic vaccine for RSV.     

Respiratory syncytial virus F envelope protein associates with lipid rafts without a requirement for other virus proteins

Like many enveloped viruses, human respiratory syncytial virus (RSV) assembles at and buds from lipid rafts. Translocation of the envelope proteins to these membrane subdomains is essential for production of infectious virus, but the targeting mechanism is poorly understood and it is not known if other virus proteins are required. Here we demonstrate that F protein of RSV intrinsically targets to lipid rafts without a requirement for any other virus protein, including the SH and G envelope proteins. Recombinant virus deficient in SH and G but retaining F protein expression was used to demonstrate that F protein still localized in rafts in both A549 and HEp-2 cells. Expression of a recombinant F gene by use of plasmid vectors demonstrated that F contains its own targeting domain and localized to rafts in the absence of other virus proteins. The domain responsible for translocation was then mapped. Unlike most other virus envelope proteins, F is unusual since the target signal is not contained within the cytoplasmic domain nor did it involve fatty acid modified residues. Furthermore, exchange of the transmembrane domain with that of the vesicular stomatitis virus G protein, a nonraft protein, did not alter F protein raft localization. Taken together, these data suggest that domains present in the extracellular portion of the protein are responsible for lipid raft targeting of the RSV F protein.     

Receptor Binding Domain of Escherichia coli F18 Fimbrial Adhesin FedF Can Be both Efficiently Secreted and Surface Displayed in a Functional Form in Lactococcus lactis

Adherence of F18 fimbrial Escherichia coli to porcine intestinal epithelial cells is mediated by the adhesin (FedF) of F18 fimbriae. In a previous study, we demonstrated the specificity of the amino acid residues between 60 and 109 as the receptor binding domain of FedF. In this study, different expression, secretion, and anchoring systems for the receptor binding domain of the FedF adhesin in Lactococcus lactis were evaluated. Two partially overlapping receptor binding domains (42 and 62 amino acid residues) were expressed as fusions with L. lactis subsp. cremoris protein PrtP for evaluation of secretion efficiency. To evaluate the cell surface display of these FedF-PrtP fusions, they were further combined with different lengths of PrtP spacers fused with either the L. lactis AcmA anchor or the PrtP cell wall binding domain. An HtrA-defective L. lactis NZ9000 mutant was constructed to determine its effect on the level of secreted or anchored fusion proteins. Recombinant L. lactis clones secreting the receptor binding domain of F18 fimbriae as a fusion with the H domains of L. lactis protein PrtP were first constructed by using two different signal peptides. FedF-PrtP fusions, directed by the signal sequence of L. brevis SlpA, were throughout found to be secreted at significantly higher quantities than corresponding fusions with the signal peptide of L. lactis Usp45. In the surface display systems tested, the L. lactis AcmA anchor performed significantly better, particularly in the L. lactis NZ9000ΔhtrA strain, compared to the L. lactis PrtP anchor region. Of the cell surface display constructs with the AcmA anchor, only those with the longest PrtP spacer regions resulted in efficient binding of recombinant L. lactis cells to porcine intestinal epithelial cells. These results confirmed that it is possible to efficiently produce the receptor binding domain of the F18 adhesin in a functionally active form in L. lactis.     

Protein F: an adhesin of Streptococcus pyogenes binds fibronectin via two distinct domains

The binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C-terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the /V-terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N-terminal domain was mixed in a binding reaction with a protein extract containing the C-terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C-terminal domain, blocked the binding partially. A clone exclusively expressing the C-terminal domain, completely blocked the binding of the 30 kDa N-terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N-terminal domain failed to block the binding of this FN fragment. Thus, the two FN-binding domains of protein F are necessary for maximal bacterial binding and act in concert to enhance the binding to fibronectin. The possibility that the two domains bind to two different regions on the fibronectin molecule is discussed.     

SED1/MFG‐E8: A Bi‐Motif protein that orchestrates diverse cellular interactions

MFG‐E8 was initially identified as a principle component of the Milk Fat Globule, a membrane‐encased collection of proteins and triglycerides that bud from the apical surface of mammary epithelia during lactation. It has since been independently identified in many species and by many investigators and given a variety of names, including p47, lactadherin, rAGS, PAS6/7, and BA‐46. The acronym SED1 was proposed to bring cohesion to this nomenclature based upon it being a Secreted protein that contains two distinct functional domains: an N‐terminal domain with two EGF‐repeats, the second of which has an integrin‐binding RGD motif, and a C‐terminal domain with two Discoidin/F5/8C domains that bind to anionic phospholipids and/or extracellular matrices. SED1/MFG‐E8 is now known to participate in a wide variety of cellular interactions, including phagocytosis of apoptotic lymphocytes and other apoptotic cells, adhesion between sperm and the egg coat, repair of intestinal mucosa, mammary gland branching morphogenesis, angiogenesis, among others. This article will explore the various roles proposed for SED1/MFG‐E8, as well as its provocative therapeutic potential. J. Cell. Biochem. 106: 957–966, 2009. © 2009 Wiley‐Liss, Inc.     

Structural differences among subfamilies of EF‐hand proteins—A view from the pseudo two‐fold symmetry axis

We have analyzed the conformations of EF‐lobes, adjacent pairs of EF‐hand domains, in a coordinate system based on the approximate two‐fold (z) axis that relates the two EF‐hands. Two parameters ‐ dE(ø), the azimuthal angle between the y‐axis and the projection of the offset vector to helix E onto the yz‐plane, and δdF(ø), the difference angle between the two helices (F1 and F2) of odd and even domains—characterize the openness of a single EF‐hand domain and of an EF‐lobe, respectively. We describe and compare values of dE(ø) and of δdF(ø) for EF‐hand proteins of five subfamilies—CTER, CPV, S100, PARV, CALP—in calci‐ and apo‐ forms, with and without bound target proteins. Each subfamily has characteristic changes associated with binding calcium and/or target proteins. Proteins 2014; 82:2915–2924. © 2014 Wiley Periodicals, Inc.     

Domain structure and conserved epitopes of Sfb protein, the fibronectin-binding adhesin of Streptococcus pyogenes

Streptococcus pyogenes expresses a fibronectin-binding surface protein (Sfb protein) which mediates adherence to human epithelial cells. The nucleotide sequence of the sfb gene was determined and the primary sequence of the Sfb protein was analysed. The protein consists of 638 amino acids and comprises five structurally distinct domains. The protein starts with an N-terminal signal peptide followed by an aromatic domain. The central part of the protein is formed by four proline-rich repeats which are flanked by non-repetitive spacer sequences. A second repeat region, consisting of four repeats that are distinct from the proline repeats and have been shown to form the fibronectin-binding domain, is located in the Cterminal part of the protein. The protein ends with a typical cell wall and membrane anchor region. Comparative sequence analysis of the N-terminal aromatic domain revealed similarities with carbohydrate-binding sites of other proteins. The proline repeat region of the Sfb protein shares characteristic features with proline-rich repeats of functionally distinct surface proteins from pathogenic Gram-positive cocci. Immunoelectron microscopy revealed an even distribution of the fibronectin-binding domain of Sfb protein on the surface of streptococcal cells. Analyses of 38 sfb genes originating from different S. pyogenes isolates revealed primary sequence variability in regions coding for the N-termini of mature Sfb proteins, whereas sequences coding for the central and C-terminal repeats were highly conserved. The repeat sequences are postulated to act as target sites for intragenic recombination events that result in variable numbers of repeats within the different sfb genes. A model of the Sfb protein is presented.     

Molecular analysis of three major wall-associated proteins of Bacillus subtilis 168: evidence for processing of the product of a gene encoding a 258 kDa precursor two-domain ligand-binding protein

Antisera raised to a 109 kDa wall-associated protein (WAP) of Bacillus subtilis 168 cross-reacts with two other WAPs of 220 and 58 kDa. The structural gene for the 109 kDa WAP (designated wapA) was cloned, sequenced, mapped at around 340° on the B. subtilis 168 chromosome and found to encode a precursor of all three wall-bound forms (2334 amino acids and 258 329 Da). The protein has two ligand-binding domains; the N-terminal domain has three direct repeats of 102 residues with 40% identity, which are responsible for wall binding. The C-terminal domain consists of two blocks of residues with a conserved motif repeated a total of 31 times. The motif consensus sequence GXXXX(Y,F)XYDXXG is almost identical to that of the Escherichia coli rearrangement hot spot family and shows similarity to a carbohydrate-binding motif of a number of Gram-positive secreted proteins. A mutant insertionally inactivated in the wapA gene had no distinguishable phenotype apart from lacking the three WAPs. The possible role of WAPA and its two-domain relationship with other ligand-binding proteins is discussed.     

Collagen‐like proteins of pathogenic streptococci

The collagen domain, which is defined by the presence of the Gly‐X‐Y triplet repeats, is amongst the most versatile and widespread known structures found in proteins from organisms representing all three domains of life. The streptococcal collagen‐like (Scl) proteins are widely present in pathogenic streptococci, including Streptococcus pyogenes, S. agalactiae, S. pneumoniae, and S. equi. Experiments and bioinformatic analyses support the hypothesis that all Scl proteins are homotrimeric and cell wall‐anchored. These proteins contain the rod‐shaped collagenous domain proximal to cell surface, as well as a variety of outermost non‐collagenous domains that generally lack predicted functions but can be grouped into one of six clusters based on sequence similarity. The well‐characterized Scl1 proteins of S. pyogenes show a dichotomous switch in ligand binding between human tissue and blood environments. In tissue, Scl1 adhesin specifically recognizes the wound microenvironment, promotes adhesion and biofilm formation, decreases bacterial killing by neutrophil extracellular traps, and modulates S. pyogenes virulence. In blood, ligands include components of complement and coagulation‐fibrinolytic systems, as well as plasma lipoproteins. In all, the Scl proteins signify a large family of structurally related surface proteins, which contribute to the ability of streptococci to colonize and cause diseases in humans and animals.     

A GLYCOPROTEIN NONCOVALENTLY ASSOCIATED WITH CELL‐WALL POLYSACCHARIDE OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

The cells of the red microalga Porphyridium sp. (UTEX 637) are encapsulated in a cell wall of a negatively charged mucilaginous polysaccharide complex composed of 10 different sugars, sulfate, and proteins. In this work, we studied the proteins associated with the cell‐wall polysaccharide. A number of noncovalently associated proteins were resolved by SDS‐PAGE, but no covalently bound proteins were detected. The most prominent protein detected was a 66‐kDa glycoprotein consisting of a polypeptide of approximately 58 kDa and a glycan moiety of approximately 8 kDa containing N‐linked terminal mannose. In size‐exclusion chromatography, the 66‐kDa protein was coeluted with the polysaccharide and could be separated from the polysaccharide only after denaturation of the protein, indicating that the 66‐kDa protein was tightly bound to the polysaccharide. Western blot analysis revealed that the 66‐kDa protein was specific to Porphyridium sp. and P. cruentum, because it was not detected in the other species of red microalgae examined. Indirect immunofluorescence assay confirmed the location of the protein in the algal cell wall. The sequence of cDNA clone encoding the 66‐kDa glycoprotein, detected in our in‐house expressed sequence tag database of Porphyridium sp., revealed that this is a novel protein with no similarity to any protein in the public domain databases and our in‐house expressed sequence tag database of the red microalga Rhodella reticulata. The 66‐kDa protein bound polysaccharides from red algae but not from those of other origins tested. Possible roles of the 66‐kDa protein in the biosynthesis of the polysaccharide are discussed.     

O glycosylation of glycoprotein G of human respiratory syncytial virus is specified within the divergent ectodomain.

cDNAs encoding the G glycoprotein of respiratory syncytial virus and the hemagglutinin-neuraminidase (HN) glycoprotein of parainfluenza virus type 3 were modified by site-specific mutagenesis and restriction fragment replacement to encode chimeric proteins consisting of the cytoplasmic and transmembrane domains of one protein fused to the ectodomain of the other. In the case of the HN ectodomain attached to the G transmembrane and cytoplasmic domains, cell surface expression of the chimera was reduced. Otherwise, the presence of the heterologous transmembrane and cytoplasmic domains had little effect on the processing of the HN or G ectodomain, as assayed by the acquisition of N-linked and O-linked carbohydrates, transport to the cell surface and, in the case of HN, folding, oligomerization, and hemadsorption activity. These results showed that the synthesis and processing of each ectodomain did not require the homologous transmembrane and cytoplasmic domains. In particular, O glycosylation of the G protein was specified fully by its ectodomain, even though this domain is highly divergent among the respiratory syncytial virus antigenic subgroups. In addition, whereas the cytoplasmic and transmembrane domains of the G protein were relatively highly conserved, they were nonetheless fully replaceable without significantly affecting processing.     

Structure of a major antigenic site on the respiratory syncytial virus fusion glycoprotein in complex with neutralizing antibody 101F

Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope ～16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.     

Evidence for the role of glycoprotein G of respiratory syncytial virus in binding of Neisseria meningitidis to HEp-2 cells

Abstract Viral glycoproteins G and F are expressed on the surface of cells infected with respiratory syncytial virus (RSV). We investigated the role of these proteins in the previously reported enhanced binding of Neisseria meningitidis to RSV-infected HEp-2 cells. Virus particles attached to bacteria were detected by immunofluorescence with flow cytometry. Binding of FITC-labelled bacteria to RSV-infected cells was significantly inhibited by monoclonal antibody against glycoprotein G. Unlabelled bacteria interfered with binding of the anti-G monoclonal antibody to these cells. These interactions were not found with a monoclonal antibody against glycoprotein F. We propose that glycoprotein G of RSV expressed on the surface of infected cells might act as an additional receptor for meningococci.     

Multiple plant RNA binding proteins identified by PCR: expression of cDNAs encoding RNA binding proteins targeted to chloroplasts in Nicotiana plumbaginifolia

Summary Pre-mRNA processing in eukaryotic cells requires the participation of multiple protein factors and ribonucleoprotein particles. One class of proteins involved in this process are RNA-binding proteins, which contain a domain of ca. 90 amino acids with a characteristic ribonucleoprotein consensus sequence (RNP-CS). A PCR approach that is suitable for the characterization of RNP-CS-type proteins is described. Fifteen different RNA-binding domains were amplified from Nicotiana tabacum (tobacco) using oligonucleotide primers specific for the sequences (K/R)G(F/Y)(G/A)FVX(F/Y) and (L/I/V)(F/Y)(V/I)(G/K)(N/G)L, which are conserved in known RNP-CS proteins. Using the tobacco domains as probes, cDNAs encoding two RNA-binding proteins, each containing two RNP-CS-type domains, were characterized in N. plumbaginifolia. The proteins, designated CP-RBP30 and CP-RBP31, are targeted to chloroplasts as demonstrated by expression of epitope-tagged cDNAs in transfected protoplasts, followed by indirect immunofluorescence. High levels of mRNA for each protein were found in leaves but not in roots, and expression of the CP-RBP31 mRNA was strongly regulated by light. The N. plumbaginifolia proteins described in this work are distinct from chloroplast RNA-binding proteins characterized recently in tobacco and spinach.     

Constitutive production of the receptor-binding domain of the F18 fimbriae on the surface of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> using synthetic promoters

Nine synthetic constitutive promoters with different activities were produced for the surface display of the receptor-binding domain of the F18 fimbrial adhesin FedF in Lactococcus lactis. The promoters were synthesized from an oligonucleotide, designed to allow for randomization of bases between defined lactococcal promoter consensus regions. However, promoters with spontaneous modifications were selected for fedF expression, indicating that the consensus promoter was too strong. The amount of FedF on the surface of the best performing promoter clone was comparable to the amount of FedF produced by a L. lactis strain with an optimized NICE expression system. Stability of FedF production further suggested that an optimal constitutive expression level was attained.     

Replication-competent or attenuated, nonpropagating vesicular stomatitis viruses expressing respiratory syncytial virus (RSV) antigens protect mice against RSV challenge

Foreign glycoproteins expressed in recombinant vesicular stomatitis virus (VSV) can elicit specific and protective immunity in the mouse model. We have previously demonstrated the expression of respiratory syncytial virus (RSV) G (attachment) and F (fusion) glycoprotein genes in recombinant VSV. In this study, we demonstrate the expression of RSV F and G glycoproteins in attenuated, nonpropagating VSVs which lack the VSV G gene (VSVDeltaG) and the incorporation of these RSV proteins into recombinant virions. We also show that intranasal vaccination of mice with nondefective VSV recombinants expressing RSV G (VSV-RSV G) or RSV F (VSV-RSV F) elicited RSV-specific antibodies in serum (by enzyme-linked immunosorbent assay [ELISA]) as well as neutralizing antibodies to RSV and afford complete protection against RSV challenge. In contrast, VSVDeltaG-RSV F induced detectable serum antibodies to RSV by ELISA, but no detectable neutralizing antibodies, yet it still protected from RSV challenge. VSVDeltaG-RSV G failed to induce any detectable serum (by ELISA) or neutralizing antibodies and failed to protect from RSV challenge. The attenuated, nonpropagating VSVDeltaG-RSV F is a particularly attractive candidate for a live attenuated recombinant RSV vaccine.