A novel and effective intranasal immunization strategy for respiratory syncytial virus.

In designing subunit vaccination strategies for respiratory syncytial virus (RSV), immunization by mucosal routes may present a realistic alternative to parenteral administration for inducing protective immune responses. To this end, we have utilized the BALB/c mouse model and an adjuvant formulation containing caprylic/capric glycerides (CCG) and polyoxyethylene-20-sorbitan monolaurate (PS). The intranasal (i.n.) delivery of purified natural F protein (3 microg per vaccine) formulated with CCG-PS resulted in the generation of statistically heightened serum anti-F protein immunoglobulin G (IgG), IgG1, IgG2b, and IgA antibodies. In addition, the presence of locally produced anti-F protein IgA was demonstrated in both vaginal and nasal washes of vaccinated mice. That production of specific serum and mucosal immunoglobulins resulted in functional immune responses was shown in neutralizing antibody assays and protection of mouse lungs against subsequent live virus challenge. Consequently, we propose a novel vaccine formulation composed of purified natural RSV F protein in CCG-PS as a viable intranasal immunogen to stimulate anti-RSV immune responses in humans.     

Respiratory syncytial virus recombinant F protein (residues 255-278) induces a helper T cell type 1 immune response in mice

We have developed and evaluated an immunodominant respiratory syncytial virus (RSV) F antigen in a mouse model. The antigenic region corresponding to amino acids 255-278 of the RSV F protein was cloned into a vector containing the ctxA(2)B gene of cholera toxin (CT). The recombinant protein was expressed in Escherichia coli and analyzed on sodium dodecyl sulfate-polyacrylamide gels. The purified protein was evaluated by immunoblot and ganglioside GM(1) enzyme-linked immunosorbent assay to confirm the expression of the RSV F protein and to correct association of the recombinant protein to form a holotoxin-like chimera, respectively. We hypothesized that genetic fusion of modified CT-based adjuvant with RSV F immunodominant epitopes (rRF-255) would induce protective humoral and cellular immune responses in mice. Intranasal immunization of mice with rRF-255 overall induced higher concentrations of anti-RSV F-specific antibodies in both serum and saliva as compared with mice immunized intranasally with RSV or phosphate-buffered saline (PBS). Antibody isotype analysis (IgA, IgG1, IgG2a, and IgG2b) was also performed. The predominant IgG2a antibody isotype response in combination with cytokine analysis of helper T cell type 1 (interferon-gamma, interleukin [IL]-2, IL-12 p70, and tumor necrosis factor-alpha) and helper T cell type 2 (IL-4 and IL-10) responses revealed that rRF-255 antigen induces a prominent helper T cell type 1 immune response in mice. The rRF-255 antigen also induced serum neutralizing antibodies in immunized mice. Analysis of RSV load in lungs showed that rRF-255 immunization provided significant protection compared with PBS control animals.     

Plasmid DNA encoding the respiratory syncytial virus G protein is a promising vaccine candidate

Respiratory syncytial virus (RSV) remains a major cause of severe respiratory diseases in infants, young children, and the elderly. However, development of a RSV vaccine has been hampered by the outcome of the infant trials in the 1960s with a formalin-inactivated RSV preparation. Enhanced lung disease was induced by the vaccination post-RSV exposure. Previous studies in mice primed with RSV G protein either formulated in adjuvants or delivered by recombinant vaccinia viruses have indicated that enhanced lung pathology resulted from a Th2-type host immune response against the viral G protein. However, in the present report, we have demonstrated that vaccination with plasmid vectors encoding either a full-length or a secreted G protein (DNA-G) clearly elicited balanced systemic and pulmonary Th1/Th2 cytokine responses in mice and did not induce an atypical pulmonary inflammatory reaction post-RSV challenge in cotton rats. DNA-G immunization also induced marked virus neutralizing antibody responses and protection against RSV infection of the lower respiratory tract of both mice and cotton rats. So far, only genetic immunization has been able to induce a balanced Th1/Th2 response with the RSV G protein, reminiscent of that induced by live RSV. Therefore, DNA-G is a promising immunogen for inclusion in a nucleic acid RSV vaccine.     

Targeting of Nasal Mucosa-Associated Antigen-Presenting Cells In Vivo with an Outer Membrane Protein A Derived from Klebsiella pneumoniae

Administration of vaccines by the nasal route has recently proven to be one of the most efficient ways for inducing both mucosal and systemic antibody responses in experimental animals. Our results demonstrate that P40, a well-defined outer membrane protein A from Klebsiella pneumoniae, is indeed a carrier molecule suitable for nasal immunization. Using fragments from the respiratory syncytial virus subgroup A (RSV-A) G protein as antigen models, it has been shown that P40 is able to induce both systemic and mucosal immunity when fused or coupled to a protein or a peptide and administered intranasally (i.n.) to naive or K. pneumoniae-primed mice. Confocal analyses of nasal mucosa-associated lymphoid tissue after i.n. instillation of P40 showed that this molecule is able to cross the nasal epithelium and target CD11c-positive cells likely to be murine dendritic cells or macrophages. More importantly, this targeting of antigen-presenting cells following i.n. immunization with a subunit of the RSV-A molecule in the absence of any mucosal adjuvant results in both upper and lower respiratory tract protection against RSV-A infection.     

The immunostimulating complex (ISCOM) is an efficient mucosal delivery system for respiratory syncytial virus (RSV) envelope antigens inducing high local and systemic antibody responses

ISCOM is an efficient mucosal delivery system for RSV envelope proteins as measured by antibody responses in respiratory tract secretions and in sera of mice following two intranasal (i.n.) administrations. Intranasally administered RSV ISCOMs induced high levels of IgA antibodies both in the upper respiratory tract and in the lungs. In the lungs, a prominent and long-lasting IgA response was recorded, which still persisted 22 weeks after the second i.n. immunization when the experiment ended. Subcutaneous (s.c.) immunization only induced low IgA titres in the upper respiratory tract and no measurable response to RSV was found in the lungs. Differences were also noticed in serum between the i.n. and s.c. modes of immunization. ISCOMs given intranasally induced earlier, higher and longer lasting IgM and IgG1 serum anti-RSV antibody responses than those induced by the s.c. mode of administration. A low serum IgE response was only detectable at 2 weeks after i.n. immunization with ISCOMs and after s.c. immunization with an inactivated virus, but no IgE response was detectable after s.c. injection of ISCOMs. The serum IgA response was more pronounced following s.c. injection of inactivated virus than after i.n. application of ISCOMs, and a clear-cut booster effect was obtained with a second immunization. Virtually no serum IgA response was detected after the s.c. administration of ISCOMs. In conclusion, the high immune responses induced by RSV ISCOMs in the respiratory tract and serum after i.n. administration indicate prominent mucosal delivery and adjuvant properties of the ISCOMs, warranting further studies.     

Immunoglobulin-A antibodies in upper airway secretions may inhibit intranasal influenza virus replication in mice but not protect against clinical illness

Mice immunized intranasally with a formalin-inactivated A/PR/8/34 (H1N1) influenza whole virus vaccine adjuvanted with cholera toxin, outer membrane vesicles from group B meningococci or formalin-inactivated whole cell Bordetella pertussis were protected against replication of the homologous virus in the nasal cavity. Only some mice were protected against clinical illness measured as weight loss and lowered body temperature. All mice immunized subcutaneously with one-tenth the intranasal vaccine dose without adjuvant were protected against clinical illness but not against local mucosal viral replication. Replicating virus was primarily found in animals with low concentrations of immunoglobulin (Ig)-A antibodies in saliva regardless of concentrations of IgG antibodies in serum. Clinical illness was seen only in those with low serum antibodies regardless of antibody levels in saliva. Nonreplicating nasal vaccines may not be sufficiently protective unless they also have a substantial influence on systemic immunity.     

Reduction of Respiratory Syncytial Virus Titer in the Lungs of Mice after Intranasal Immunization with a Chimeric Peptide Consisting of a Single CTL Epitope Linked to a Fusion Peptide

In the work described here, the effect of intranasal immunization of BALB/c mice with synthetic chimeric peptides consisting of a cytotoxic T-cell epitope (amino acids 81–95) from the M2 protein of respiratory syncytial virus (RSV) and a fusion peptide (amino acids 113–131) from the F1 protein of measles virus on response to challenge with RSV has been assessed. Three intranasal immunizations with the chimeric peptides without adjuvant induce peptide- and RSV-specific cytotoxic T-cell responses (CTL) at 1 or 3 weeks after the third immunization. The CTL responses significantly declined at 6 weeks after immunization. Furthermore, viral load in the lungs following challenge with RSV was significantly reduced in mice immunized with the F/M2:81-95 chimeric peptide compared to control animals at 1 or 3 weeks after immunization and no reduction of RSV titers was detectable 6 weeks after immunization. The CTL activity induced by F/M2:81-95 was therefore short-lived (less than 6 weeks) but was significantly correlated with the reduction in viral load in the lungs.     

Comparison of inactivated, live and recombinant DNA vaccines against influenza virus in a mouse model

The protective efficacy of influenza hemagglutinin expressed from recombinant vaccinia virus was compared with that induced by inactivated or infectious influenza vaccines. Intraperitoneal and intranasal routes of vaccination were compared. All the vaccines except the intranasally administered, inactivated vaccine induced detectable levels of neutralizing and hemagglutination-inhibiting antibodies in the serum of mice at 28 days postvaccination. Immunization with any of the intranasally administered vaccines reduced the amount of influenza virus nucleoprotein antigen in lungs after challenge with a homologous, mouse-adapted strain of influenza virus. Intraperitoneally administered vaccines failed to provide such protection. These results indicated that the route of vaccine administration may be the most critical factor for inducing protective immunity. The results also showed that in this mouse model a recombinant DNA-based vaccine could provide protection equivalent to that provided by conventional attenuated and inactivated influenza vaccines.     

Induction of antibody responses in the common mucosal immune system by respiratory syncytical virus immunostimulating complexes

Immunostimulating complexes (ISCOMs) containing envelope proteins of respiratory syncytial virus (RSV) were explored as a mucosal delivery system for the capacity of inducing a common mucosal antibody response. Two intranasal (i.n.) administrations of BALB/c mice with ISCOMs induced potent serum IgG, and strong IgA responses to RSV locally in the lungs and the upper respiratory, and remotely in the genital and the intestinal tracts. Virtually no measurable IgA response was found in these mucosal organs after two subcutaneous (s.c.) immunizations. Virus neutralizing (VN) antibodies were detected in serum and in all of the mucosal organ extracts after both s.c. and i.n. immunizations indicating that the neutralizing epitopes were preserved after both mucosal and parenteral modes of administration. While the mucosal IgA response appears to be of mucosal origin, the IgG antibodies to RSV detected in the mucosal organs were likely of serum origin. However, the mucosal VN antibodies correlated with the IgG rather than the IgA levels. An enhanced IgA response to gp120 in various mucosal organs was recorded after i.n. immunization with gp120 incorporated in RSV ISCOMs, indicating a role of RSV envelope proteins in enhancing and targeting mucosal responses to passenger antigens. Received: 23 October 1998     

Chimeric subgroup A respiratory syncytial virus with the glycoproteins substituted by those of subgroup B and RSV without the M2-2 gene are attenuated in African green monkeys

Using the existing reverse genetics system developed for the subgroup A respiratory syncytial virus (RSV), a chimeric virus (designated rA-G(B)F(B)) that expresses subgroup B-specific antigens was constructed by replacing the G and F genes of the A2 strain with those of the 9320 strain of subgroup B RSV. rA-G(B)F(B) grew well in tissue culture, but it was attenuated in the respiratory tracts of cotton rats and African green monkeys. To further attenuate this chimeric RSV, the M2-2 open reading frame was removed from rA-G(B)F(B). rA-G(B)F(B)DeltaM2-2 was highly attenuated in replication in the respiratory tracts of the infected monkeys, but it provided complete protection against wild-type subgroup B RSV challenge following two doses of infection. In this study, rA2DeltaM2-2 (a recombinant A2 RSV that lacks the M2-2 gene) was also evaluated in African green monkeys. The replication of rA2DeltaM2-2 was highly restricted in both the upper and lower respiratory tracts of the infected monkeys and it induced titers of serum anti-RSV neutralizing antibody that were slightly lower than those induced by wild-type rA2. When rA2DeltaM2-2-infected monkeys were challenged with wild-type A2 virus, the replication of the challenge virus was reduced by approximately 100-fold in the upper respiratory tract and 45,000-fold in the lower respiratory tracts. rA2DeltaM2-2 and rA-G(B)F(B)DeltaM2-2 could represent a bivalent RSV vaccine composition for protection against multiple strains from the two RSV subgroups.     

Rapid recovery in mice after combined nasal/oral immunization with killed respiratory syncytial virus

Based on the concept of a common mucosal immune system, the murine gastrointestinal tract was inoculated (oral) with three doses (5, 20, and 40 micrograms) of UV-inactivated respiratory syncytial virus (RSV) in order to elicit a virus-specific immune response in the respiratory tract. Only the 40 micrograms dose induced significant (P less than 0.01) anti-RSV-IgG rises in serum and lung wash compared to controls. To improve the immune response, mice were immunized intranasally under light anesthesia with the same 40 micrograms dosage regimen of killed RSV so that each dose passed through the nose and was swallowed. This combined nasal/oral immunization stimulated anti-RSV-IgG in serum, lung wash and nasal wash (P less than 0.001) and anti-RSV-IgA in lung and nasal wash (P less than 0.001) that were comparable to levels after infection with live RSV. Three days after challenge with live RSV, mice given combined nasal/oral immunization showed suppressed nasal virus shedding (P = 0.025). Nasal virus shedding correlated inversely with concentrations of anti-RSV-Ig in nasal secretions but did not correlate with concentrations in serum.     

Erysipelothrix rhusiopathiae YS-1 as a Live Vaccine Vehicle for Heterologous Protein Expression and Intranasal Immunization of Pigs

We have developed a system in which a foreign antigen is delivered and expressed on the surface of an attenuated strain of Erysipelothrix rhusiopathiae YS-1 and have examined the ability of a such recombinant E. rhusiopathiae strain to function as a mucosal vaccine vector. The C-terminal portion, including two repeat regions, R1 and R2, of the P97 adhesin of Mycoplasma hyopneumoniae strain E-1 was successfully translocated and expressed on the E. rhusiopathiae YS-1 cell surface after it was fused to SpaA.1, a cell surface protective antigen of E. rhusiopathiae. BALB/c mice subcutaneously immunized with the E. rhusiopathiae recombinant strains developed specific antibodies against SpaA.1 protein and were protected from lethal challenge with the highly virulent homologous E. rhusiopathiae Fujisawa-SmR strain, showing the efficacy of this heterologous-antigen expression system as a vaccine against E. rhusiopathiae infection. To determine whether protective immune responses are induced in target species, newborn, specific-pathogen-free piglets were immunized intranasally with a recombinant strain designated YS-19. The immunized piglets developed specific anti-SpaA.1 immunoglobulin G (IgG) antibodies in their serum and were protected from death by erysipelas, showing that mucosal vaccination of piglets with YS-19 induces systemic immune responses. Furthermore, YS-19-immunized piglets showed higher levels of P97-specific IgA antibodies in the respiratory tract than did YS-1-immunized piglets. Thus, E. rhusiopathiae YS-1 appears to be a promising vaccine vector for mucosal delivery that can induce local and systemic immune responses.     

Intranasal immunization with pneumococcal conjugate vaccines with LT-K63, a nontoxic mutant of heat-Labile enterotoxin, as adjuvant rapidly induces protective immunity against lethal pneumococcal infections in neonatal mice

Immunization with pneumococcal polysaccharides (PPS) conjugated to tetanus toxoid (TT) (Pnc-TT) elicits protective immunity in an adult murine pneumococcal infection model. To assess immunogenicity and protective immunity in early life, neonatal (1 week old) and infant (3 weeks old) mice were immunized intranasally (i.n.) or subcutaneously (s.c.) with Pnc-TT of serotype 1 (Pnc1-TT). Anti-PPS-1 and anti-TT immunoglobulin G (IgG) and IgM antibodies were measured in serum and saliva, and vaccine-induced protection was evaluated by i.n. challenge with serotype 1 pneumococci. Pnc1-TT was immunogenic in neonatal and infant mice when administered s.c. without adjuvant: a majority of the young mice were protected from bacteremia and a reduction of pneumococcal density in the lungs was observed, although antibody responses and protective efficacy remained lower than in adults. The addition of LT-K63, a nontoxic mutant of heat-labile enterotoxin, as adjuvant significantly enhanced PPS-1-specific IgG responses and protective efficacy following either s.c. or i.n. Pnc1-TT immunization. Mucosal immunization was particularly efficient in neonates, as a single i.n. dose of Pnc1-TT and LT-K63 induced significantly higher PPS-1-specific IgG responses than s.c. immunization and was sufficient to protect neonatal mice against pneumococcal infections, whereas two s.c. doses were required to induce complete protection. In addition, i.n. immunization with Pnc1-TT and LT-K63 induced a vigorous salivary IgA response. This suggests that mucosal immunization with pneumococcal conjugate vaccines and LT-K63 may be able to circumvent some of the limitations of neonatal antibody responses, which are required for protective immunity in early life.     

Effects of alveolar macrophage depletion on liposomal vaccine protection against respiratory syncytial virus (RSV)

Little is known about the identities and roles of antigen-presenting cells upon exposure to antigens of respiratory syncytial virus (RSV). Here, we focused on elucidating the importance of alveolar macrophages in conferring protective immunity in mice administered a liposome-encapsulated recombinant fragment of the RSV G protein. Mice were depleted of alveolar macrophages by intranasal inoculation of liposome-encapsulated dichloromethylenediphosphonic acid (DMDP). Mice depleted of alveolar macrophages prior to immunization developed reduced levels of serum RSV-neutralizing antibody and showed dramatically impaired protection against RSV challenge. The severity of interstitial inflammation was also markedly reduced in macrophage-depleted mice. In conclusion, this study demonstrates a pivotal role for alveolar macrophages during exposure to liposome-encapsulated RSV antigen in initiating both protective and histopathological responses against RSV.     

Immunogenicity and protection studies with recombinant mycobacteria and vaccinia vectors coexpressing the 18-kilodalton protein of Mycobacterium leprae.

The activation of antigen-specific T lymphocytes is essential for the control of leprosy infection in humans and experimental animals. T cells recognize a variety of protein antigens from Mycobacterium leprae, including the 18-kDa protein, which is limited in distribution among mycobacteria and which is absent from Mycobacterium tuberculosis and the vaccine strain, Mycobacterium bovis BCG. Adjuvant preparations of mycobacterial protein antigens have had limited protective efficacy for experimental infections in animals. Since recombinant vectors may elicit more effective T-cell responses than adjuvant preparations, recombinant vaccinia virus (VV18) and M. bovis BCG (BCG18) vectors expressing the 18-kDa protein of M. leprae were prepared. Both VV18 and BCG18 stimulated anti-18-kDa protein antibody and lymphocyte proliferative responses. Sequential immunization with VV18 followed by BCG18 induced higher levels of specific immunoglobulin G2a antibodies than immunoglobulin G1 antibodies, in contrast to immunization with VV18 or BCG18 alone. The protective efficacy of immunization with VV18 from a challenge with BCG18 was examined in two murine models of mycobacterial infection. After intravenous challenge, mice immunized with recombinant vaccinia virus exhibited lower initial levels of replication and earlier clearance of BCG18 from their spleens than mice immunized with vaccinia virus expressing an unrelated protein. After footpad infection in a dissemination model, there was earlier clearance of BCG18 from specifically immunized mice. However, immunization of mice with VV18 did not prevent a productive mycobacterial infection.     

Protection abilities of influenza B virus DNA vaccines expressing hemagglutinin, neuraminidase, or both in mice

Every year, a vaccination against Influenza B virus (IBV) is essential due to an antigenic variation. Development of an efficient and convenient vaccine is important for the prevention of viral infection. This study reports examination of the protective immunity in mice evoked by a single inoculation of plasmid DNA expressing hemagglutinin (HA DNA) or neuraminidase (NA DNA) of IBV. The HA DNA or NA DNA was injected intramuscularly into BALB/c mice separately or as a mixture. The injection of plasmid was followed by an electroporation close to the site of puncture. Four weeks later, the immunized mice were challenged with a lethal dose of IBV. The protective abilities of DNA vaccines were evaluated by the detection of specific antibodies in serum, survival rate, virus titer in lungs, and change of body weight. We found that a single dose of HA DNA or NA DNA induced the formation of specific antibodies and conferred effective protection against the lethal challenge of IBV. However, the combined vaccine HA DNA and NA DNA enhanced the protective ability of immunized mice. The obtained results suggested that immunization with single dose of HA DNA, NA DNA or with combination of both could be an efficient method for preventing IBV infection. Key words: DNA vaccine; Influenza B virus; hemagglutinin; neuraminidase.     

Antibodies to the central conserved region of respiratory syncytial virus (RSV) G protein block RSV G protein CX3C-CX3CR1 binding and cross-neutralize RSV A and B strains

Respiratory syncytial virus (RSV) is a primary cause of severe lower respiratory tract disease in infants, young children, and the elderly worldwide, and despite decades of effort, there remains no safe and effective vaccine. RSV modifies the host immune response during infection by CX3C chemokine mimicry adversely affecting pulmonary leukocyte chemotaxis and CX3CR1+ RSV-specific T-cell responses. In this study we investigated whether immunization of mice with RSV G protein polypeptides from strain A2 could induce antibodies that block G protein-CX3CR1 interactions of both RSV A and B strains. The results show that mice immunized with RSV A2 G polypeptides generate antibodies that block binding of RSV A2 and B1 native G proteins to CX3CR1, and that these antibodies effectively cross-neutralize both A and B strains of RSV. These findings suggest that vaccines that induce RSV G protein-CX3CR1 blocking antibodies may provide a disease intervention strategy in the efforts to develop safe and efficacious RSV vaccines.     

Characteristics of immunity induced by viral antigen or conferred by antibody via different administration routes

The characteristics of the immunity induced by viral antigens or conferred by antiviral antibody via different routes of administration were evaluated comparatively. C57BL/6 mice were immunized via intranasal, intradermal or enteric routes with a live recombinant vaccinia virus expressing the respiratory syncytial virus (RSV) F glycoprotein (F.rVV) or RSV, and then challenged intranasally with RSV. Inhibition of RSV replication was observed in the lungs of all the mice; however, only intranasal immunization hindered virus replication in the nose. Lung inflammation, characterized by infiltration of neutrophils and of mononuclear cells was strongest in the intradermally immunized mice, but was observed in all F.rVV immunized mice to various degrees. Intranasal administration of a potently neutralizing human anti-RSV antibody Fab fragment to infected mice inhibited RSV replication in the nose and, when combined with intraperitoneal administration, protected both the lung and the nose in the absence of deleterious lung pathology. These data suggest that intranasal immunization with F.rVV reduces RSV replication in the respiratory tract, but still induces pathological lung inflammation, even though this is milder than that observed following intradermal immunization. Local neutralizing antibody is indispensable for protection in the nose.