Structural characterization of archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) formulations prepared by different protocols and their efficacy upon intranasal immunization of mice

Intranasal administration of ovalbumin (OVA) formulated in an archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) system prepared by the addition of CaCl2 to small unilamellar archaeosomes (liposomes made from archaeal polar lipids) containing encapsulated OVA, was recently shown to elicit strong and sustained OVA-specific mucosal and systemic immune responses. In this study, we show that the centrifugation/washing and antigen quantization steps required in the standard protocol for obtaining OVA/AMVAD model vaccine formulations can be eliminated by using simpler protocols such as admixing OVA with preformed empty archaeosomes, or by changing the starting ratio (w/w) of archaeal lipid to antigen at the archaeosome preparation stage, prior to the addition of CaCl2 to convert to the AMVAD structures. Irrespective of the vaccine preparation protocol, the AMVAD particle typically comprised of larger spherical structures that had aggregated like a bunch of grapes, and it contained aqueous compartment(s). The anti-OVA IgA antibody responses in vaginal wash, nasal wash, serum, and bile samples, and the anti-OVA IgG antibody responses in sera, in mice intranasally immunized with the OVA/AMVAD formulations prepared by the simplified or the standard protocols, were comparable.     

Intranasal immunization with a recombinant truncated FimH adhesin adjuvanted with CpG oligodeoxynucleotides protects mice against uropathogenic Escherichia coli challenge

In this work, we assessed the efficacy of an experimental intranasal vaccine against urinary-tract infections. The vaccine contained a recombinant truncated FimH (rFimHt) adhesin plus CpG oligodeoxynucleotides. The efficacy of the vaccine was compared with that of an intramuscular vaccine that was formulated with the same immunogen plus Freund's adjuvant. Our results show that serum immunoglobulin G titers of vaccinated animals were similarly enhanced in both cases. However, the intranasal vaccine elicited higher vaginal-wash-specific immunoglobulin A titers against rFimHt than the intramuscular route. Both vaccines reduced the in vivo colonization of the bladder by uropathogenic Escherichia coli more than 100-fold in a murine cystitis model. Our results indicate that a recombinant truncated FimH adhesin plus CpG oligodeoxynucleotides is a suitable immunogenic combination that can contribute to the development of a highly efficacious urinary tract infection vaccine.     

Intranasal immunization of guinea pigs with an immunodominant foot-and-mouth disease virus peptide conjugate induces mucosal and humoral antibodies and protection against challenge

Guinea pigs immunized intranasally with a keyhole limpet hemocyanin-linked peptide, corresponding to the prominent G-H loop of the VP1 protein of foot-and-mouth disease virus, raised substantial levels of antipeptide and virus-neutralizing antibodies in sera and of peptide-specific secretory immunoglobulin A in nasal secretions. In groups of animals immunized intranasally without adjuvant, 86 percent were fully protected upon challenge with homotypic virus. Surprisingly, animals given the peptide conjugates plus the mucosal adjuvant cholera toxin were afforded only partial protection in that primary lesions were observed in most animals, although spread to other feet was prevented. These results indicate that intranasal inoculation with the peptide offers a potential route of vaccination against foot-and-mouth disease and may be useful for eliciting protection in the upper respiratory tracts of susceptible animals.     

Intranasal immunization with recombinant toxin-coregulated pilus and cholera toxin B subunit protects rabbits against Vibrio cholerae O1 challenge

Intranasal immunization, a noninvasive method of vaccination, has been found to be effective in inducing systemic and mucosal immune responses. The present study was aimed at investigating the efficacy of intranasal immunization in inducing mucosal immunity in experimental cholera by subunit recombinant protein vaccines from Vibrio cholerae O1. The structural genes encoding toxin-coregulated pilus A (TcpA) and B subunit of cholera toxin (CtxB) from V. cholerae O1 were cloned and expressed in Escherichia coli . Rabbits were immunized intranasally with purified TcpA and CtxB alone or a mixture of TcpA and CtxB. Immunization with TcpA and CtxB alone conferred, respectively, 41.1% and 70.5% protection against V. cholerae challenge, whereas immunization with a mixture of both antigens conferred complete (100%) protection, as assayed in the rabbit ileal loop model. Serum titers of immunoglobulin G (IgG) antibodies to TcpA and CtxB, and anti-TcpA- and anti-CtxB-specific sIgA in intestinal lavage of vaccinated animals were found to be significantly elevated compared with unimmunized controls. Vibriocidal antibodies were detected at remarkable levels in rabbits receiving TcpA antigen and their titers correlated with protection. Thus, mucosal codelivery of pertinent cholera toxoids provides enhanced protection against experimental cholera.     

An antivector vaccine protects against a lethal vector-borne pathogen

Vaccines that target blood-feeding disease vectors, such as mosquitoes and ticks, have the potential to protect against the many diseases caused by vector-borne pathogens. We tested the ability of an anti-tick vaccine derived from a tick cement protein (64TRP) of Rhipicephalus appendiculatus to protect mice against tick-borne encephalitis virus (TBEV) transmitted by infected Ixodes ricinus ticks. The vaccine has a "dual action" in immunized animals: when infested with ticks, the inflammatory and immune responses first disrupt the skin feeding site, resulting in impaired blood feeding, and then specific anti-64TRP antibodies cross-react with midgut antigenic epitopes, causing rupture of the tick midgut and death of engorged ticks. Three parameters were measured: "transmission," number of uninfected nymphal ticks that became infected when cofeeding with an infected adult female tick; "support," number of mice supporting virus transmission from the infected tick to cofeeding uninfected nymphs; and "survival," number of mice that survived infection by tick bite and subsequent challenge by intraperitoneal inoculation of a lethal dose of TBEV. We show that one dose of the 64TRP vaccine protects mice against lethal challenge by infected ticks; control animals developed a fatal viral encephalitis. The protective effect of the 64TRP vaccine was comparable to that of a single dose of a commercial TBEV vaccine, while the transmission-blocking effect of 64TRP was better than that of the antiviral vaccine in reducing the number of animals supporting virus transmission. By contrast, the commercial antitick vaccine (TickGARD) that targets only the tick's midgut showed transmission-blocking activity but was not protective. The 64TRP vaccine demonstrates the potential to control vector-borne disease by interfering with pathogen transmission, apparently by mediating a local cutaneous inflammatory immune response at the tick-feeding site.     

Evaluation in rhesus macaques of Tat and rev-targeted immunization as a preventive vaccine against mucosal challenge with SHIV-BX08

Recent evidence suggests that a CD8-mediated cytotoxic T-cell response against the regulatory proteins of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) may control infection after pathogenic virus challenge. Here, we evaluated whether vaccination with Tat or Tat and Rev could significantly reduce viral load in nonhuman primates. Rhesus macaques were primed with Semliki forest Virus (SFV) expressing HIV-1 tat (SFV-tat) and HIV-1 rev (SFV-rev) and boosted with modified vaccinia virus Ankara (MVA) expressing tat and rev. A second group of monkey was primed with SFV-tat only and boosted with MVA-tat. A third group received a tat and rev DNA/MVA prime-boost vaccine regimen. Monitoring of anti-Tat and anti-Rev antibody responses or antigen-specific IFN-gamma production, as measured by enzyme-linked immunospot assays revealed no clear differences between the three groups. These results suggest that priming with either DNA or SFV seemed to be equivalent, but the additive or synergistic effect of a rev vaccine could not be clearly established. The animals were challenged by the rectal route 9 weeks after the last booster immunization, using 10 MID(50) of a SHIV-BX08 stock. Postchallenge follow-up of the monkeys included testing seroconversion to Gag and Env antigens, measuring virus infectivity in PBMC by cocultivation with noninfected human cells, and monitoring of plasma viral load. None of the animals was protected from infection as assessed by PCR, but peak viremia was reduced more than 200-fold compared to sham controls in one third (6/18) of vaccinated macaques, whatever the vaccine regimen they received. Interestingly, among these six protected animals four did not seroconvert. Altogether, these results clearly indicated that the addition of early HIV proteins like Tat and Rev in a multicomponent preventive vaccine including structural proteins like Env or Gag may be beneficial in preventive vaccinal strategies.     

Intranasal immunization with temperature-sensitive mutants protects granulocytopenic mice from lethal pulmonary challenge withPseudomonas aeruginosa

Intranasal (i.n.) immunization with two temperature-sensitive (ts) mutants ofPseudomonas aeruginosa protected, in a dose-related manner, granulocytopenic (GCP) mice challenged with a lethal dose of the wild-type (wt) organism. The number of ts mutants in oronasopharyngeal lavage fluids and stools decreased steadily in both normal and GCP mice after i.n. immunization. Intranasal immunization with 10⁷ colony-forming units (CFU) of either mutant induced significant protection, whereas intraperitoneal (i.p.) immunization with similar doses induced lower protection. Protection induced by i.n. immunization was accompanied by increased levels of anti-P. aeruginosa IgA in lung lavage fluids. The results of this study demonstrate the usefulness of ts mutants ofP. aeruginosa for local immunization to protect GCP hosts from fatalP. aeruginosa pneumonia.     

Immunoprotective activity and safety of a respiratory syncytial virus vaccine: mucosal delivery of fusion glycoprotein with a CpG oligodeoxynucleotide adjuvant

CpG oligodeoxynucleotides (ODN) were identified that stimulated immunoglobulin production and cell proliferation in cotton rat cells in vitro. Three of these ODN were used as a mucosal adjuvant in the noses of cotton rats immunized via this route with respiratory syncytial virus fusion (F) protein. The CpG ODN markedly increased the cotton rat humoral neutralizing-antibody response to respiratory syncytial virus. Such immunized animals had a marked reduction in the production of infectious virus after a live-virus challenge. Animals immunized with the combination of F protein and CpG developed enhanced pulmonary pathology consisting of alveolitis and interstitial pneumonitis after a live-virus challenge. Similar enhanced disease has been seen in cotton rats and children immunized with formalin-inactivated respiratory syncytial virus.     

Intranasal immunization with H5N1 vaccine plus Poly I:Poly C12U, a Toll-like receptor agonist, protects mice against homologous and heterologous virus challenge

The avian H5N1 influenza virus has the potential to cause a new pandemic. Since it is difficult to predict which strain of influenza will cause a pandemic, it is advantageous to produce vaccines that confer cross-protective immunity. Mucosal vaccine administration was reported to induce cross-protective immunity by inducing secretion of IgA at the mucosal surface. Adjuvants can also enhance the development of fully protective mucosal immunity. Here we show that a new mucosal adjuvant, poly I:poly C12U (Ampligen), a Toll-like receptor 3 agonist proven to be safe in a Phase III human trial, is an effective adjuvant for H5N1 influenza vaccination. Intranasal administration of a candidate influenza vaccine with Ampligen resulted in secretion of IgA, and protected mice that were subsequently challenged with homologous A/Vietnam/1194/2004 and heterologous A/HK/483/97 and A/Indonesia/6/2005 virus.     

A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge.

A candidate live-virus vaccine strain of Venezuelan equine encephalitis virus (VEE) was configured as a replication-competent vector for in vivo expression of heterologous immunogens. Three features of VEE recommend it for use as a vaccine vector. (i) Most human and animal populations are not already immune to VEE, so preexisting immunity to the vector would not limit expression of the heterologous antigen. (ii) VEE replicates first in local lymphoid tissue, a site favoring the induction of an effective immune response. (iii) Parenteral immunization of rodents and humans with live, attenuated VEE vaccines protects against mucosal challenge, suggesting that VEE vaccine vectors might be used successfully to protect against mucosal pathogens. Upon subcutaneous (s.c.) inoculation into the footpad of mice, a VEE vector containing the complete influenza virus hemagglutinin (HA) gene expressed HA in the draining lymph node and induced anti-HA immunoglobulin G (IgG) and IgA serum antibodies, the levels of which could be increased by s.c. booster inoculation. When immunized mice were challenged intranasally with a virulent strain of influenza virus, replication of challenge virus in their lungs was restricted, and they were completely protected from signs of disease. Significant reduction of influenza virus replication in the nasal epithelia of HA vector-immunized mice suggested an effective immunity at the mucosal surface. VEE vaccine vectors represent an alternative vaccination strategy when killed or subunit vaccines are ineffective or when the use of a live attenuated vaccine might be unsafe.     

A formalin inactivated whole SIVmac vaccine in Ribi adjuvant protects against homologous and heterologous SIV challenge.

Eight monkeys were immunized at 0, 4, 9, and 18 weeks with a total of 2 mg of formalin inactivated SIVmac vaccine with Ribi adjuvant. Two weeks after the last booster four immunized monkeys and two controls were challenged with 10 MID50 of live homologous virus SIVmac, and the remaining four vaccinated animals along with two controls were challenged with the heterologous SIVsm strain. All eight vaccinated monkeys resisted the virus challenge, whereas all controls became infected. Three months after the first challenge the monkeys were rechallenged with the same virus strain, without further boosting. Two of four vaccinated monkeys were still resistant to the homologous SIV strain, and three of four monkeys were resistant to the heterologous SIVsm strain. This study demonstrates vaccine induced cross-protection between SIV strains.     

Intranasal administration of a detoxified endotoxin vaccine protects mice against heterologous Gram-negative bacterial pneumonia

When given passively or elicited actively, antibodies induced by a detoxified Escherichia coli J5 mutant lipopolysaccharide (J5dLPS)-group B meningococcal outer membrane protein (-OMP) vaccine previously protected animals from lethal sepsis. To assess the use of this vaccine for the treatment of Gram-negative bacillary pneumonia, we vaccinated mice, with or without the adjuvant CpG, by intranasal (i.n.) or intraperitoneal (i.p.) routes of administration. Local and systemic IgG levels were 2-3 logs higher following i.p. immunization compared to i.n. However, i.n. immunization elicited both local and systemic IgA, unlike i.p. administration. The addition of CpG to the vaccine, by either route of administration, elicited greater levels of antibody. Intranasal immunization protected mice against lethal heterologous Gram-negative bacillary pneumonia and post-immunization serum and broncho-alveolar lavage fluid mediated enhanced bacterial killing with peritoneal and alveolar macrophages in vitro. We conclude that further studies on the use of J5dLPS-OMP for the prevention of nosocomial pneumonia are warranted.     

Vesicular stomatitis virus-based vaccine protects hamsters against lethal challenge with Andes virus

Andes virus (ANDV) is a highly pathogenic South American hantavirus that causes hantavirus pulmonary syndrome (HPS). A high case fatality rate, the potential for human-to-human transmission, the capacity to infect via aerosolization, and the absence of effective therapies make it imperative that a safe, fast-acting, and effective ANDV vaccine be developed. We generated and characterized a recombinant vesicular stomatitis virus (VSV) vector expressing the ANDV surface glycoprotein precursor (VSVΔG/ANDVGPC) as a possible vaccine candidate and tested its efficacy in the only lethal-disease animal model of HPS. Syrian hamsters immunized with a single injection of VSVΔG/ANDVGPC were fully protected against disease when challenged at 28, 14, 7, or 3 days postimmunization with a lethal dose of ANDV; however, the mechanism of protection seems to differ depending on when the immunization occurs. At 28 days postimmunization, a lack of detectable ANDV RNA in lung, liver, and blood tissue samples, as well as a lack of seroconversion to the ANDV nucleocapsidprotein in nearly all animals, suggested largely sterile immunity. The vaccine was able to generate high levels of neutralizing anti-ANDV G(N)/G(C) antibodies, which seem to play a role as a mechanism of vaccine protection. Administration of the vaccine at 7 or 3 days before challenge also resulted in full protection but with no specific neutralizing humoral immune response, suggesting a possible role of innate responses in protection against challenge virus replication. Administration of the vaccine 24 h postchallenge was successful in protecting 90% of hamsters and again suggested the induction of a potent antiviral state by the recombinant vector as a potential mechanism. Overall, our data suggest the potential for the use of the VSV platform as a fast-acting and effective prophylaxis/postexposure treatment against lethal hantavirus infections.     

Modified vaccinia virus Ankara protects macaques against respiratory challenge with monkeypox virus

The use of classical smallpox vaccines based on vaccinia virus (VV) is associated with severe complications in both naive and immune individuals. Modified vaccinia virus Ankara (MVA), a highly attenuated replication-deficient strain of VV, has been proven to be safe in humans and immunocompromised animals, and its efficacy against smallpox is currently being addressed. Here we directly compare the efficacies of MVA alone and in combination with classical VV-based vaccines in a cynomolgus macaque monkeypox model. The MVA-based smallpox vaccine protected macaques against a lethal respiratory challenge with monkeypox virus and is therefore an important candidate for the protection of humans against smallpox.     

DNA immunization with plasmids encoding fusion and nucleocapsid proteins of bovine respiratory syncytial virus induces a strong cell-mediated immunity and protects calves against challenge

Respiratory syncytial viruses (RSV) are one of the most important respiratory pathogens of humans and cattle, and there is currently no safe and effective vaccine prophylaxis. In this study, we designed two codon-optimized plasmids encoding the bovine RSV fusion (F) and nucleocapsid (N) proteins and assessed their immunogenicity in young calves. Two administrations of both plasmids elicited low antibody levels but primed a strong cell-mediated immunity characterized by lymphoproliferative response and gamma interferon production in vitro and in vivo. Interestingly, this strong cellular response drastically reduced viral replication, clinical signs, and pulmonary lesions after a highly virulent challenge. Moreover, calves that were further vaccinated with a killed-virus vaccine developed high levels of neutralizing antibody and were fully protected following challenge. These results indicate that DNA vaccination could be a promising alternative to the classical vaccines against RSV in cattle and could therefore open perspectives for vaccinating young infants.     

Lactococcus lactis as an adjuvant and delivery vehicle of antigens against pneumococcal respiratory infections

Most studies of Lactococcus lactis as delivery vehicles of pneumococcal antigens are focused on the effectiveness of mucosal recombinant vaccines against Streptococcus pneumoniae in animal models. At present, there are three types of pneumococcal vaccines: capsular polysaccharide pneumococcal vaccines (PPV), protein-polysaccharide conjugate pneumococcal vaccines (PCV) and protein-based pneumococcal vaccines (PBPV). Only PPV and PCV have been licensed. These vaccines, however, do not represent a definitive solution. Novel, safe and inexpensive vaccines are necessary, especially in developing countries. Probiotic microorganisms such as lactic acid bacteria (LAB) are an interesting alternative for their use as vehicles in pneumococcal vaccines due to their GRAS (Generally Recognized As Safe) status. Thus, the adjuvanticity of Lactococcus lactis by itself represents added value over the use of other bacteria, a question dealt with in this review. In addition, the expression of different pneumococcal antigens as well as the use of oral and nasal mucosal routes of administration of lactococcal vaccines is considered. The advantages of nasal live vaccines are evident; nonetheless, oral vaccines can be a good alternative when the adequate dose is used. Another point addressed here is the use of live versus inactivated vaccines. In this sense, few researchers have focused on inactivated strains to be used as vaccines against pneumoccoccus. The immunogenicity of live vaccines is better than the one afforded by inactivated ones; however, the probiotic-inactivated vaccine combination has improved this matter considerably. The progress made so far in the protective immune response induced by recombinant vaccines, the successful trials in animal models and the safety considerations of their application in humans suggest that the use of recombinant vaccines represents a good short-term option in the control of pneumococcal diseases.     

Intranasal immunization with CpG oligodeoxynucleotides as an adjuvant dramatically increases IgA and protection against herpes simplex virus-2 in the genital tract

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.     

Intranasal immunization with chitosan microparticles enhances LACK-DNA vaccine protection and induces specific long-lasting immunity against visceral leishmaniasis

Development of a protective vaccine against Leishmania depends on antigen formulation and adjuvants that induce specific immunity and long-lasting immune responses. We previously demonstrated that BALB/c mice intranasally vaccinated with a plasmid DNA encoding the p36/LACK leishmanial antigen (LACK-DNA) develop a protective immunity for up to 3 months after vaccination, which was linked with the systemic expression of vaccine mRNA in peripheral organs. In this study, LACK-DNA vaccine was associated with biocompatible chitosan microparticles cross-linked with glyceraldehyde (CMC) to boost the long-lasting immunity against the late Leishmania infantum challenge. Infection at 7 days, 3 or 6 months after vaccination resulted in significantly lower parasite loads when compared with non-vaccinated controls. Besides, LACK-DNA-chitosan vaccinated mice showed long-time protection observed after the late time point challenge. The achieved protection was correlated with an enhanced spleen cell responsiveness to parasite antigens, marked by increased proliferation and IFN-γ as well as decreased IL-10 production. Moreover, we found diminished systemic levels of TNF-α that was compatible with the better health condition observed in LACK-DNA/CMC vaccinated-infected mice. Together, our data indicate the feasibility of chitosan microparticles as a delivery system tool to extend the protective immunity conferred by LACK-DNA vaccine, which may be explored in vaccine formulations against Leishmania parasite infections.     

Intranasal delivery of cationic PLGA nano/microparticles-loaded FMDV DNA vaccine encoding IL-6 elicited protective immunity against FMDV challenge

Mucosal vaccination has been demonstrated to be an effective means of eliciting protective immunity against aerosol infections of foot and mouth disease virus (FMDV) and various approaches have been used to improve mucosal response to this pathogen. In this study, cationic PLGA (poly(lactide-co-glycolide)) nano/microparticles were used as an intranasal delivery vehicle as a means administering FMDV DNA vaccine encoding the FMDV capsid protein and the bovine IL-6 gene as a means of enhancing mucosal and systemic immune responses in animals. Three eukaryotic expression plasmids with or without bovine IL-6 gene (pc-P12A3C, pc-IL2AP12A3C and pc-P12AIL3C) were generated. The two latter plasmids were designed with the IL-6 gene located either before or between the P12A and 3C genes, respectively, as a means of determining if the location of the IL-6 gene affected capsid assembly and the subsequent immune response. Guinea pigs and rats were intranasally vaccinated with the respective chitosan-coated PLGA nano/microparticles-loaded FMDV DNA vaccine formulations. Animals immunized with pc-P12AIL3C (followed by animals vaccinated with pc-P12A3C and pc-IL2AP12A3C) developed the highest levels of antigen-specific serum IgG and IgA antibody responses and the highest levels of sIgA (secretory IgA) present in mucosal tissues. However, the highest levels of neutralizing antibodies were generated in pc-IL2AP12A3C-immunized animals (followed by pc-P12AIL3C- and then in pc-P12A3C-immunized animals). pc-IL2AP12A3C-immunized animals also developed stronger cell mediated immune responses (followed by pc-P12AIL3C- and pc-P12A3C-immunized animals) as evidenced by antigen-specific T-cell proliferation and expression levels of IFN-γ by both CD4+ and CD8+ splenic T cells. The percentage of animals protected against FMDV challenge following immunizations with pc-IL2AP12A3C, pc-P12AIL3C or pc-P12A3C were 3/5, 1/5 and 0/5, respectively. These data suggested that intranasal delivery of cationic PLGA nano/microparticles loaded with various FMDV DNA vaccine formulations encoding IL-6 as a molecular adjuvant enhanced protective immunity against FMDV, particularly pc-IL2AP12A3C with IL-6 gene located before P12A3C gene.     

A single intranasal inoculation with a paramyxovirus-vectored vaccine protects guinea pigs against a lethal-dose Ebola virus challenge

To determine whether intranasal inoculation with a paramyxovirus-vectored vaccine can induce protective immunity against Ebola virus (EV), recombinant human parainfluenza virus type 3 (HPIV3) was modified to express either the EV structural glycoprotein (GP) by itself (HPIV3/EboGP) or together with the EV nucleoprotein (NP) (HPIV3/EboGP-NP). Expression of EV GP by these recombinant viruses resulted in its efficient incorporation into virus particles and increased cytopathic effect in Vero cells. HPIV3/EboGP was 100-fold more efficiently neutralized by antibodies to EV than by antibodies to HPIV3. Guinea pigs infected with a single intranasal inoculation of 10(5.3) PFU of HPIV3/EboGP or HPIV3/EboGP-NP showed no apparent signs of disease yet developed a strong humoral response specific to the EV proteins. When these animals were challenged with an intraperitoneal injection of 10(3) PFU of EV, there were no outward signs of disease, no viremia or detectable EV antigen in the blood, and no evidence of infection in the spleen, liver, and lungs. In contrast, all of the control animals died or developed severe EV disease following challenge. The highly effective immunity achieved with a single vaccine dose suggests that intranasal immunization with live vectored vaccines based on recombinant respiratory viruses may be an advantageous approach to inducing protective responses against severe systemic infections, such as those caused by hemorrhagic fever agents.