Cell-penetrating peptides for transmucosal delivery of proteins.

Enabling non-invasive delivery of proteins across the mucosal barriers promises improved patient compliance and therapeutic efficacies. Cell-penetrating peptides (CPPs) are emerging as a promising and versatile tool to enhance protein and peptide permeation across various mucosal barriers. This review examines the structural and physicochemical attributes of the nasal, buccal, sublingual, and oral mucosa that hamper macromolecular delivery. Recent development of CPPs for overcoming those mucosal barriers for protein delivery is summarized and analyzed. Perspectives regarding current challenges and future research directions towards improving non-invasive transmucosal delivery of macromolecules for ultimate clinical translation are discussed.

In vivo tracing of immunostimulatory raw starch microparticles after mucosal administration.

Raw starch microparticles (SMPs) proved efficient antigen carriers with adjuvant properties when administered via the mucosal route; however, the underlying mechanisms associated with this bioactivity are unknown. In the present study, we explored the mucoadhesion properties, fate, and toxicity of starch microparticles after mucosal administration. Nasally administered microparticles were mainly retained in nasal turbinates, reaching the nasal-associated lymphoid tissue; this step is facilitated by the ability of the microparticles to penetrate through the mucous epithelium. Likewise, we found intraduodenally administered SMPs on the small intestinal villi, follicle-associated epithelium, and Peyer's patches. Furthermore, under simulated gastric and intestinal pH conditions, we detected mucoadhesion between the SMPs and mucins, regardless of microparticle swelling. SMPs' mucoadhesion and translocation to mucosal immune responses induction sites explain the previously reported role of these microparticles as vaccine adjuvants and immunostimulants.

Mucosal administration of anti-bacterial antibodies provide long-term cross-protection against Pseudomonas aeruginosa respiratory infection.

Bacterial respiratory infections, either acute or chronic, are major threats to human health. Direct mucosal administration, through the airways, of therapeutic antibodies (Abs) offers a tremendous opportunity to benefit patients with respiratory infections. The mode of action of anti-infective Abs relies on pathogen neutralization and crystallizable fragment (Fc)-mediated recruitment of immune effectors to facilitate their elimination. Using a mouse model of acute pneumonia induced by Pseudomonas aeruginosa, we depicted the immunomodulatory mode of action of a neutralizing anti-bacterial Abs. Beyond the rapid and efficient containment of the primary infection, the Abs delivered through the airways harnessed genuine innate and adaptive immune responses to provide long-term protection, preventing secondary bacterial infection. In vitro antigen-presenting cells stimulation assay, as well as in vivo bacterial challenges and serum transfer experiments indicate an essential contribution of immune complexes with the Abs and pathogen in the induction of the sustained and protective anti-bacterial humoral response. Interestingly, the long-lasting response protected partially against secondary infections with heterologous P. aeruginosa strains. Overall, our findings suggest that Abs delivered mucosally promotes bacteria neutralization and provides protection against secondary infection. This opens novel perspectives for the development of anti-infective Abs delivered to the lung mucosa, to treat respiratory infections.

Evaluation of Oral Transmucosal Administration of Pentobarbital for Euthanasia of Conscious Wild Birds.

This prospective study evaluated oral transmucosal pentobarbital sodium at three doses in 110 wild-caught wild birds requiring euthanasia. Birds received transmucosal pentobarbital at five (430 mg/kg), six (516 mg/kg), and seven times (602 mg/kg) the intravenous dose for mammals. Time to first effects and loss of consciousness, presence of pupillary light and corneal reflexes, apnea, and asystole were recorded each minute. When asystole was not achieved at 5 minutes, IV pentobarbital was administered. Combining data for all doses, loss of consciousness occurred at a median (range) of 2 minutes (0-4.75 min), apnea at 3 minutes (0-6 min), and asystole at 4 minutes (0.5-5 min). Loss of consciousness and apnea occurred significantly faster in the 602 mg/kg dose group than in the 430 mg/kg group (p = 0.009, difference of 0.6 ± 0.2 min; p = 0.024, difference of 0.7 ± 0.3 min), respectively. Apnea and asystole were achieved in 80/110 birds within 5 minutes. Oral transmucosal pentobarbital results in rapid loss of consciousness and respiratory arrest and provides a reliable alternative euthanasia method compared to intravenous administration.

Estudio observacional, prospectivo y multicéntrico sobre el uso de citrato de fentanilo transmucosa en la práctica clínica habitual para el control de las crisis de dolor irruptivo en pacientes oncológicos / Observational, prospective and multicenter study on the use of transmucosal fentanyl citrate in regular clinical practice for the control of breakthrough pain crises in cancer patients

Objetivo: Evaluar el grado de satisfacción de los pacientes y/o cuidadores con el tratamiento de citrato de fentanilo transmucosa en la gestión de las crisis de dolor irruptivo oncológico en términos de facilidad de uso. Métodos: Se realizó un estudio observacional, prospectivo y multicéntrico con 48 pacientes sometidos a tratamiento de mantenimiento con opioides para el dolor crónico basal provocado por el cáncer y que, además, sufrían crisis de dolor irruptivo para las que estaban recibiendo tratamiento con citrato de fentanilo. La variable principal del estudio fue el grado de satisfacción de los pacientes y/o sus cuidadores con el citrato de fentanilo en el manejo de las crisis de dolor irruptivo oncológico, evaluadas mediante cuestionarios Escala Visual Analógica (EVA). Resultados: El 90,6 % de los pacientes/cuidadores muestran un elevado grado de satisfacción con el empleo de citrato de fentanilo en términos de facilidad de uso (valor medio EVA de 8,2). Por su parte, tanto la valoración general por parte de los pacientes/cuidadores como por parte de los sanitarios ha sido muy positiva (valor medio EVA 7,7). Conclusiones: El citrato de fentanilo es una terapia fácil de usar y eficaz para el tratamiento de las crisis de dolor irruptivo oncológico, con amplia aceptación tanto por parte de pacientes y cuidadores como de los profesionales sanitarios.(AU)

Objective: To assess the degree of satisfaction of patients and/or caregivers with transmucosal fentanyl citrate treatment in the management of breakthrough cancer pain crises in terms of ease of use. Methods: An observational, prospective and multicenter study was carried out with 48 patients undergoing maintenance treatment with opioids for baseline chronic cancer pain and who, in addition, suffered breakthrough pain crises for those who were receiving treatment with fentanyl citrate. The main variable of the study was the degree of satisfaction of the patients and/or their caregivers with fentanyl citrate in the management of breakthrough cancer pain crises, evaluatedby means of Visual Analogue Scale (VAS) questionnaires. Results: 90.6 % of patients/caregivers show a high degree of satisfaction with the use of fentanyl citrate in terms of ease of use (mean VAS value of 8.2). For its part, both the general assessment by the patients/caregivers and by the healthcare professionals has been very positive (mean VAS value 7.7). Conclusions: Fentanyl citrate is an easy-to-use and effective therapy for the treatment of breakthrough cancer pain crises, widely accepted by both patients and caregivers as well as health professionals.(AU)

Oromucosal products - Market landscape and innovative technologies: A review.

Strategies that rely on oral mucosal administration have increased in the last decade and oromucosal products are paving the way to overcome specific challenges, namely improving drug bioavailability when compared with the conventional oral route, due to a reduction of the hepatic first-pass metabolism and pre-systemic degradation. Overall, the advantages of these products make oromucosal route of administration attractive for the development of value-added medicines, which can address more properly the unmet medical needs of specific patients. Generally, such products have an easy and convenient administration since they do not require water for ingestion, which may be particularly relevant for geriatric and pediatric groups, or non-cooperative patients. Usually, the development of these products aims to provide a faster onset of action, critical for acute or emergency treatments. Although oriented to achieve better therapeutic outcomes, today's drug development is primarily focused on patient-centered care, meaning that patients' specific characteristics/needs are an important driving force behind product-development efforts. In accordance, pharmaceutical innovation can rely not only on new drug substances but also on re-formulation of already approved ones or alternative routes of administration, enhancing patient convenience, treatment efficacy and/or safety. Throughout this review, the oromucosal drug products, approved in the last decade, and a retrospective analysis of their critical quality attributes and specifications will be described. Furthermore, trends and opportunities of the latest technologies in this field, as well as the number of ongoing clinical studies, will be presented and discussed.

Pharmacokinetics of intravenous, oral transmucosal, and intranasal buprenorphine in healthy male dogs.

Effective management of pain in animals is of critical importance but options are limited for treating acute pain in dogs on an outpatient basis. The objective of this study was to compare the plasma concentrations and pharmacokinetics of a concentrated solution of buprenorphine, 1.8 mg/ml (Simbadol™) administered intravenously, intranasally, and via the oral transmucosal (OTM) route in healthy male dogs. Five healthy castrated adult male Beagle-cross dogs were included in this randomized blocked crossover study. The dogs received 0.03 mg/kg body weight buprenorphine intravenously, intranasally, or via the OTM route, with a minimum 72-h washout period between treatments. Blood samples were collected at multiple intervals up to 24 h post administration and buprenorphine plasma concentrations were determined by liquid chromatography tandem mass spectrometry. Non-compartmental pharmacokinetic analysis revealed that the area under the curve of intravenous, intranasal, and OTM routes were 28.0 (15.1-41.3) h × ng/ml, 16.1 (3.4-28.7) h × ng/ml and 10.8 (8.8-11.8) h × ng/ml, respectively. The bioavailability of intranasal and OTM routes were 57.5 (22.7-93.7)% and 41.1 (25.5-69.4)%, respectively. Intranasal and OTM routes of administration of concentrated buprenorphine in dogs may allow for the provision of analgesic care at home.

Immunoadjuvant and Humoral Immune Responses of Garlic (Allium sativum L.) Lectins upon Systemic and Mucosal Administration in BALB/c Mice.

Dietary food components have the ability to affect immune function; following absorption, specifically orally ingested dietary food containing lectins can systemically modulate the immune cells and affect the response to self- and co-administered food antigens. The mannose-binding lectins from garlic (Allium sativum agglutinins; ASAs) were identified as immunodulatory proteins in vitro. The objective of the present study was to assess the immunogenicity and adjuvanticity of garlic agglutinins and to evaluate whether they have adjuvant properties in vivo for a weak antigen ovalbumin (OVA). Garlic lectins (ASA I and ASA II) were administered by intranasal (50 days duration) and intradermal (14 days duration) routes, and the anti-lectin and anti-OVA immune (IgG) responses in the control and test groups of the BALB/c mice were assessed for humoral immunogenicity. Lectins, co-administered with OVA, were examined for lectin-induced anti-OVA IgG response to assess their adjuvant properties. The splenic and thymic indices were evaluated as a measure of immunomodulatory functions. Intradermal administration of ASA I and ASA II had showed a four-fold and two-fold increase in anti-lectin IgG response, respectively, vs. the control on day 14. In the intranasal route, the increases were 3-fold and 2.4-fold for ASA I and ASA II, respectively, on day 50. No decrease in the body weights of animals was noticed; the increases in the spleen and thymus weights, as well as their indices, were significant in the lectin groups. In the adjuvanticity study by intranasal administration, ASA I co-administered with ovalbumin (OVA) induced a remarkable increase in anti-OVA IgG response (~six-fold; p < 0.001) compared to the control, and ASA II induced a four-fold increase vs. the control on day 50. The results indicated that ASA was a potent immunogen which induced mucosal immunogenicity to the antigens that were administered intranasally in BALB/c mice. The observations made of the in vivo study indicate that ASA I has the potential use as an oral and mucosal adjuvant to deliver candidate weak antigens. Further clinical studies in humans are required to confirm its applicability.

Mannose-anchored quaternized chitosan/thiolated carboxymethyl chitosan composite NPs as mucoadhesive carrier for drug delivery.

There are various challenges for the mucosal delivery of drug, which is largely attributed to the absence of effective drug carriers that can make delivery to mucosal sites. In the present study, we aimed to synthesize bifunctional mucoadhesive nanoparticles (NPs) that could be used for mucosal delivery. N-2-Hydroxypropyl trimethyl ammonium chloride chitosan (M-N-2-HACC) was modified with D-mannose, and N-acetyl-L-cysteine (NAC) was immobilized on the carboxymethyl chitosan (N-CMCS). The electrostatic interaction between the two substances was used to produce mannose-modified thiolated chitosan NPs (M-N-2-HACC/N-CMCS NPs). The NPs showed a particle size of 196.72 ± 0.45 nm and zeta potential of 17.12 ± 0.50 mV. Moreover, it demonstrated high hydrophilicity, enduring drug release, stability, safety, and mucosal adhesion, which contributed to the effectiveness of mucosal administration. Additionally, the NPs could be instantly absorbed by macrophages. Collectively, these results suggested that M-N-2-HACC/N-CMCS NPs could be used as a promising candidate for mucosal delivery.

Development of novel mucoadhesive gels containing nanoparticle for buccal administration of dexamethasone

Abstract This study aimed to develop promising and innovative mucoadhesive gel systems containing dexamethasone-loaded nanoparticle to increase the effectiveness of treatment for oral precancerous lesions and to reduce side effects. In this respect, a dexamethasone-loaded nanoparticle formulation was prepared by using emulsification/solvent evaporation method. The nanoparticle has high zeta potential (-10.3±0.5 mV), low particle size (218.42±2.1), low polydispersity index (0.070±0.014) and high encapsulation efficiency (95.018±2.982%). To improve the mucosal retention time, the dexamethasone-loaded nanoparticle was dispersed in mucoadhesive gel using gellan gum. The developed gels offered appropriate pH value, high drug content, suitable mechanical and mucoadhesive performance and appropriate viscosity for mucosal administration. All formulations exhibited plastic flow and typical gel-type mechanical spectra after the determined frequency value. The developed formulations exhibited extended drug release as intended for these systems. Cytotoxicity was tested by MTT assay in human epithelioid carcinoma cell (HeLa) in vitro. The MTT assay showed that the blank formulations were non-toxic to cells. It was observed that the bioactivity of the free dexamethasone was potentiated by mucoadhesive gels containing dexamethasone-loaded nanoparticle in HeLa cells. Results from this study indicate that mucoadhesive gels are effective for the local treatment of precancerous lesions. Our findings showed that the developed formulations were worthy of further studies.

Pharmacokinetic Characterisation and Comparison of Bioavailability of Intranasal Fentanyl, Transmucosal, and Intravenous Administration through a Three-Way Crossover Study in 24 Healthy Volunteers.

BACKGROUND: For more than 60 years, the synthetic opioid fentanyl has been widely used in anaesthesia and analgesia. While the intravenous formulation is primarily used for general anaesthesia and intensive care settings, the drug's high lipophilic properties also allow various noninvasive routes of administration. Published data suggest that intranasal administration is also attractive for use as intranasal patient-controlled analgesia (PCA). A newly developed intranasal fentanyl formulation containing 47 µg fentanyl, intravenous fentanyl, and oral transmucosal fentanyl citrate were characterised, and bioavailability was compared to assess the suitability of the intranasal formulation for an intranasal PCA product. METHODS: 27 healthy volunteers were enrolled in a single-centre, open-label, randomised (order of treatments), single-dose study in a three-period crossover design. The pharmacokinetics of one intranasal puff of fentanyl formulation (47 µg, 140 mL per puff), one short intravenous infusion of 50 µg fentanyl, and one lozenge with an integrated applicator (200 µg fentanyl) were studied, and bioavailability was calculated. Blood samples were collected over 12 hours, and plasma concentrations of fentanyl were determined by HPLC with MS/MS detection. RESULTS: 24 volunteers completed the study. The geometric mean of AUC0-tlast was the highest with oral transmucosal administration (1106 h ∗ pg/ml, CV% = 32.86), followed by intravenous (672 h ∗ pg/ml, CV% = 32.18) and intranasal administration (515 h ∗ pg/ml, CV% = 30.10). C max was 886 pg/ml (CV% = 59.38) for intravenous, 338 pg/ml (CV% = 45.61) for intranasal, and 310 pg/ml (CV% = 29.58) for oral transmucosal administration. t max was shortest for intravenous administration (0.06 h, SD = 0.056), followed by intranasal (0.21 h, SD = 0.078) and oral transmucosal administration (1.20 h, SD = 0.763). Dose-adjusted absolute bioavailability was determined to be 74.70% for the intranasal formulation and 41.25% for the oral transmucosal product. In total, 38 adverse events (AEs) occurred. Fourteen AEs were potentially related to the investigational items. No serious AE occurred. CONCLUSION: Pharmacokinetic parameters and bioavailability of the investigated intranasal fentanyl indicated suitability for its intended use as an intranasal PCA option.

The Bacterial Mucosal Immunotherapy MV130 Protects Against SARS-CoV-2 Infection and Improves COVID-19 Vaccines Immunogenicity.

COVID-19-specific vaccines are efficient prophylactic weapons against SARS-CoV-2 virus. However, boosting innate responses may represent an innovative way to immediately fight future emerging viral infections or boost vaccines. MV130 is a mucosal immunotherapy, based on a mixture of whole heat-inactivated bacteria, that has shown clinical efficacy against recurrent viral respiratory infections. Herein, we show that the prophylactic intranasal administration of this immunotherapy confers heterologous protection against SARS-CoV-2 infection in susceptible K18-hACE2 mice. Furthermore, in C57BL/6 mice, prophylactic administration of MV130 improves the immunogenicity of two different COVID-19 vaccine formulations targeting the SARS-CoV-2 spike (S) protein, inoculated either intramuscularly or intranasally. Independently of the vaccine candidate and vaccination route used, intranasal prophylaxis with MV130 boosted S-specific responses, including CD8+-T cell activation and the production of S-specific mucosal IgA antibodies. Therefore, the bacterial mucosal immunotherapy MV130 protects against SARS-CoV-2 infection and improves COVID-19 vaccines immunogenicity.

Mucosal Administration of Recombinant Baculovirus Displaying Toxoplasma gondii ROP4 Confers Protection Against T. gondii Challenge Infection in Mice.

Pathogens require physical contact with the mucosal surface of the host organism to initiate infection and as such, vaccines eliciting both mucosal and systemic immune responses would be promising. Studies involving the use of recombinant baculoviruses (rBVs) as mucosal vaccines are severely lacking despite their inherently safe nature, especially against pathogens of global importance such as Toxoplasma gondii. Here, we generated rBVs displaying T. gondii rhoptry protein 4 (ROP4) and evaluated their protective efficacy in BALB/c mice following immunization via intranasal (IN) and oral routes. IN immunization with the ROP4-expressing rBVs elicited higher levels of parasite-specific IgA antibody responses compared to oral immunization. Upon challenge infection with a lethal dose of T. gondii ME49, IN immunization elicited significantly higher parasite-specific antibody responses in the mucosal tissues such as intestines, feces, vaginal samples, and brain than oral immunization. Marked increases in IgG and IgA antibody-secreting cell (ASC) responses were observed from intranasally immunized mice. IN immunization elicited significantly enhanced induction of CD4+, CD8+ T cells, and germinal center B (GC B) cell responses from secondary lymphoid organs while limiting the production of the inflammatory cytokines IFN-γ and IL-6 in the brain, all of which contributed to protecting mice against T. gondii lethal challenge infection. Our findings suggest that IN delivery of ROP4 rBVs induced better mucosal and systemic immunity against the lethal T. gondii challenge infection compared to oral immunization.

Methods to improve the immunogenicity of plasmid DNA vaccines.

DNA vaccines have emerged as innovative approaches that have great potential to overcome the limitations of current conventional vaccines. Plasmid DNA vaccines are often safer than other vaccines because they carry only antigen genetic information, are more stable and easier to produce, and can stimulate both humoral and cellular immune responses. Although the results of ongoing clinical trials are very promising, some limitations compromise the immunogenicity of these vaccines. Thus, this review describes different strategies that can be explored to improve the immunogenicity of plasmid DNA vaccines, including the optimization of the plasmid vector backbone, the use of different methods for vaccine delivery, the use of alternative administration routes and the inclusion of adjuvants. In combination, these improvements could lead to the successful clinical use of plasmid DNA vaccines.

Nanotechnologies for the delivery of biologicals: Historical perspective and current landscape.

Biological macromolecule-based therapeutics irrupted in the pharmaceutical scene generating a great hope due to their outstanding specificity and potency. However, given their susceptibility to degradation and limited capacity to overcome biological barriers new delivery technologies had to be developed for them to reach their targets. This review aims at analyzing the historical seminal advances that shaped the development of the protein/peptide delivery field, along with the emerging technologies on the lead of the current landscape. Particularly, focus is made on technologies with a potential for transmucosal systemic delivery of protein/peptide drugs, followed by approaches for the delivery of antigens as new vaccination strategies, and formulations of biological drugs in oncology, with special emphasis on mAbs. Finally, a discussion of the key challenges the field is facing, along with an overview of prospective advances are provided.

Size shifting of solid lipid nanoparticle system triggered by alkaline phosphatase for site specific mucosal drug delivery.

We aim to prepare a size-shifting nanocarrier for site-targeting mucosal drug delivery that can penetrate through mucus gel layer and remain close to the absorption membrane. As nanocarriers can be engineered to penetrate mucus but they can also back diffuse into outer mucus regions, a size shifting to micron range once they have reached the absorption membrane would prevent back-diffusion effect and extend drug release over a long period of time. For this purpose, we loaded solid lipid nanoparticles (SLN) with a phosphate ester surfactant and octadecylamine. Alkaline phosphatase (AP), a membrane bound enzyme was for the first time utilized as an in situ partner for triggering the size conversion at epithelial cell surface. Having the size of ~120 nm, SLN with hydrophilic and phosphate-decorated shells were shown to penetrate through mucus gel and form aggregates above cell layer surface. Aggregates of 5-8 µm were formed due to interparticle interactions induced by enzymatic phosphate removal after ~30 min in contact with isolated AP. The developed SLN system could be a potential tool for mucosal drug delivery to AP-expressing tissues like colon, lung, cervix, vagina and some mucus-secreting tumors.

Plasmid Replicons for the Production of Pharmaceutical-Grade pDNA, Proteins and Antigens by Lactococcus lactis Cell Factories.

The Lactococcus lactis bacterium found in different natural environments is traditionally associated with the fermented food industry. But recently, its applications have been spreading to the pharmaceutical industry, which has exploited its probiotic characteristics and is moving towards its use as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. Additionally, due to its food-grade and generally recognized safe status, there have been an increasing number of studies about its use in live mucosal vaccination. In this review, we critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. A plasmid vector is an easily customized component when the goal is to engineer bacteria in order to produce a heterologous compound in industrially significant amounts, as an alternative to genomic DNA modifications. The additional burden to the cell depends on plasmid copy number and on the expression level, targeting location and type of protein expressed. For live mucosal vaccination applications, besides the presence of the necessary regulatory sequences, it is imperative that cells produce the antigen of interest in sufficient yields. The cell wall anchored antigens had shown more promising results in live mucosal vaccination studies, when compared with intracellular or secreted antigens. On the other side, engineering L. lactis to express membrane proteins, especially if they have a eukaryotic background, increases the overall cellular burden. The different alternative replicons for live mucosal vaccination, using L. lactis as the DNA vaccine carrier or the antigen producer, are critically reviewed, as a starting platform to choose or engineer the best vector for each application.

Mucosal Delivery of Recombinant Vesicular Stomatitis Virus Vectors Expressing Envelope Proteins of Respiratory Syncytial Virus Induces Protective Immunity in Cotton Rats.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract (LRT) infections, with increased severity in high-risk human populations, such as infants, the immunocompromised, and the elderly. Although the virus was identified more than 60 years ago, there is still no licensed vaccine available. Over the years, several vaccine delivery strategies have been evaluated. In this study, we developed two recombinant vesicular stomatitis virus (rVSV) vector-based vaccine candidates expressing the RSV-G (attachment) protein (rVSV-G) or F (fusion) protein (rVSV-F). All vectors were evaluated in the cotton rat animal model for their in vivo immunogenicity and protective efficacy against an RSV-A2 virus challenge. Intranasal (i.n.) delivery of rVSV-G and rVSV-F together completely protected the lower respiratory tract (lungs) at doses as low as 103 PFU. In contrast, doses greater than 106 PFU were required to protect the upper respiratory tract (URT) completely. Reimmunization of RSV-immune cotton rats was most effective with rVSV-F. In immunized animals, overall antibody responses were sufficient for protection, whereas CD4 and CD8 T cells were not necessary. A prime-boost immunization regimen increased both protection and neutralizing antibody titers. Overall, mucosally delivered rVSV-vector-based RSV vaccine candidates induce protective immunity and therefore represent a promising immunization regimen against RSV infection.IMPORTANCE Even after decades of intensive research efforts, a safe and efficacious RSV vaccine remains elusive. Expression of heterologous antigens from rVSV vectors has demonstrated several practical and safety advantages over other virus vector systems and live attenuated vaccines. In this study, we developed safe and efficacious vaccine candidates by expressing the two major immunogenic RSV surface proteins in rVSV vectors and delivering them mucosally in a prime-boost regimen. The main immune parameter responsible for protection was the antibody response. These vaccine candidates induced complete protection of both the upper and lower respiratory tracts.