Intranasal immunization of mice with recombinant lipidated P2086 protein reduces nasal colonization of group B Neisseria meningitidis

Neisseria meningitidis is a major cause of bacterial meningitis in the human population, especially among young children. There is a need to develop a non-capsular vaccine to prevent meningococcal B infections due to the inadequate immune response elicited against the capsular polysaccharide of these strains. Previously, we developed a Swiss Webster adult mouse intranasal challenge model for group B N. meningitidis and evaluated several potential vaccine candidates including a meningococcal outer membrane protein, P2086, through parenteral immunization. Since N. meningitidis is a respiratory pathogen, a mucosal immune response may play an important role in the defense against meningococcal infections. Thus, intranasal immunization may be more effective than traditional parenteral immunization. In this study, mice were immunized intranasally with purified recombinant lipidated P2086 protein (rLP2086) adjuvanted with either CT-E29H, a genetically modified cholera toxin that is significantly reduced in enzymatic activity and toxicity or RC529-AF, a synthetic immunostimulant molecule in aqueous formulation. rLP2086-specific serum and mucosal IgG and IgA antibodies were induced. IgG antibodies reacted with whole cells of multiple strains of group B N.meningitidis. The antibodies have functional activity against N. meningitidis as demonstrated by bactericidal assays. Moreover, immunized mice exhibited reduced nasal colonization of group B meningococcal strains in the intranasal challenge model. These results demonstrate that an intranasal immunization with rLP2086 protein formulated with a detoxified cholera toxin or RC529-AF could prevent the initial colonization of group B meningococcus and become an effective immunization strategy against group B N. meningitidis.     

Intranasal immunization with phosphorylcholine induces antigen specific mucosal and systemic immune responses in mice

Phosphorylcholine (PC) is a structural component of a wide variety of pathogens including Streptococcus pneumoniae and Haemophilus influenzae, and anti-PC immune responses are known to protect mice against invasive bacterial diseases. The present study tested the capability of PC as an intranasal plurispecific vaccine against upper airway infections. BALB/c mice immunized with intranasal PC-keyhole limpet hemocyanin (KLH) plus cholera toxin (CT) as a mucosal adjuvant showed increased PC-specific IgM in serum, IgA in nasal wash and saliva, and numbers of PC-specific nasal and splenic antibody producing cells. Enhanced production of IL-4 and IFN-gamma by CD4+ T cells indicated the participation of Th2- and Th1-type cells. Salivary IgA antibodies produced by intranasal immunization with PC-KLH plus CT reacted to most strains of S. pneumoniae and H. influenzae. Further we demonstrated that the clearance of S. pneumoniae and H. influenzae from the nasal tract was significantly enhanced by nasal immunization with PC-KLH and CT. Thus, intranasal vaccination to induce PC-specific immune responses might help to prevent upper airway infections caused by S. pneumoniae and H. influenzae.     

Intranasal immunization with a replication-deficient adenovirus vector expressing glycoprotein H of murine cytomegalovirus induces mucosal and systemic immunity

A vaccine vector, designated AdV-gH, was constructed by inserting the complete open reading frame of MCMV gH under control of the human CMV IE-1 promoter into the E-1 region of a replication-deficient adenovirus 5. In vitro infection of QB1-293 cells and mouse embryo cells with AdV-gH resulted in expression of MCMV gH detected by IFA. Immunization of BALB/c mice with AdV-gH (1 x 10(7) PFU) given by the intranasal route induced a humoral response with antibody detected in serum of 100% of vaccines. Antibody to MCMV gH was also detected in the bronchoalveolar lavage, fecal suspensions and vaginal washings. The viral titer of lung and salivary gland of immunized mice 10 days after intranasal challenge with MCMV (1 x 10(5) PFU) were significantly reduced compared to controls, but virus infection was not prevented. Re-exposure of mice to AdV-gH 30 days after primary immunization induced a significant boost of serum antibody response. When rechallenged with MCMV intranasally, these mice had further reduction of MCMV titers in the lung and salivary glands. Such a strategy may be important in reducing horizontal transmission of CMV infections across mucosal surfaces and in altering host immunity to CMV.     

Immunization with recombinant Streptococcus gordonii expressing tetanus toxin fragment C confers protection from lethal challenge in mice

Tetanus toxin fragment C (TTFC) was expressed on the surface of the vaccine vector Streptococcus gordonii, a Gram-positive commensal bacterium of the human oral cavity. The immunogenicity of recombinant S. gordonii expressing TTFC was assayed in mice immunized by the parenteral and mucosal routes. High serum TTFC-specific IgG responses were induced in both BALB/c and C57BL/6 mice immunized subcutaneously. A total of 82% of vaccinated BALB/c mice were protected from the lethal challenge with 50 LD(50) of tetanus toxin (TT) and a direct correlation between the serum TTFC-specific IgG concentration and survival time of unprotected animals was observed. Intranasal immunization of BALB/c mice was also effective in inducing TTFC-specific serum IgG and local IgA in lung washes. Furthermore, 38% of animals immunized intranasally were protected from the lethal challenge with 10 LD(50) of TT while all control animals died within 24 h. Analysis of the serum IgG subclasses showed that the IgG1 subclass was predominant after parenteral immunization in BALB/c mice (IgG1/IgG2a ratio congruent with6) while following mucosal immunization a mixed IgG1 and IgG2a pattern (IgG1/IgG2a ratio congruent with1) was observed. These data show that TTFC expressed on the surface of S. gordonii is immunogenic by the subcutaneous and mucosal routes and the immune response induced is capable of conferring protection from the lethal challenge with TT.     

Intranasal immunization with inactivated SARS-CoV (SARS-associated coronavirus) induced local and serum antibodies in mice

SARS-CoV (severe acute respiratory syndrome-associated coronavirus) strain GZ50 was partially purified and inactivated with 1:2000 formaldehyde. In cell culture the inactivated virus blocked the replication of live virus by decreasing the TCID(5.0) of the live virus 10(3.6) to 10(4.6) times. Inactivated GZ50 was used to immunize mice intranasally either alone, or after precipitation with polyethylene glycol (PEG), or with CpG, or CTB as an adjuvant. The titer of serum neutralizing antibodies was up to 1:640. In mice immunized with adjuvants or PEG precipitated GZ50, specific IgA was detected in tracheal-lung wash fluid by immunofluorescence. Though serum antibodies were detected, no anti-SARS-IgA could be detected in mice immunized only with inactivated GZ50. The roles of adjuvants in intranasal immunization with inactivated. SARS-CoV is discussed.     

Nasal immunization of mice with a rotavirus DNA vaccine that induces protective intestinal IgA antibodies

DNA vaccination using a plasmid encoding the rotavirus inner capsid VP6 has been explored in the mouse model of rotavirus infection. BALB/c mice were immunized with a VP6 DNA vaccine by the intramuscular, nasal and oral routes. VP6 DNA vaccination by the nasal and oral routes induced the production of anti-VP6 IgA antibodies by intestinal lymphoid cells. Intramuscular DNA injection stimulated the production of serum anti-VP6 IgG but not serum anti-VP6 IgA antibodies. Protection against shedding of rotaviruses in stools after oral challenge with the murine EDIM rotavirus strain was investigated in the immunized mice. A significant reduction in the level of rotavirus antigen shedding was demonstrated in those mice immunized at mucosal surfaces, both orally and nasally, with the VP6 DNA vaccine. Intramuscular DNA immunization, which elicited serum anti-VP6 IgG responses but not virus-specific intestinal IgA antibodies, did not provide significant protection against rotavirus challenge.     

Intranasal immunization with recombinant PspA fused with a flagellin enhances cross-protective immunity against Streptococcus pneumoniae infection in mice

Streptococcus pneumoniae is a major respiratory pathogen that causes high levels of mortality and morbidity in infants and the elderly. Despite the use of antibiotics and vaccines, fatal pneumococcal disease remains prevalent. Pneumococcal surface protein A (PspA), a highly immunogenic surface protein produced by all strains of S. pneumoniae, can elicit protective immunity against fatal pneumococcal infection. We have previously demonstrated that the Vibrio vulnificus FlaB, a bacterial flagellin protein and agonist of TLR5, has strong mucosal adjuvant activity and induces protective immunity upon co-administration with tetanus toxoid. In this study, we have tested whether intranasal immunization with recombinant fusion proteins consisted of PspA and FlaB (PspA-FlaB and FlaB-PspA) is able to elicit more efficient protective mucosal immune responses against pneumococcal infection than immunization with PspA alone or with a stoichiometric mixture of PspA and FlaB. When mice were intranasally immunized with fusion proteins, significantly higher levels of anti-PspA IgG and IgA were induced in serum and mucosal secretions. The mice immunized intranasally with the FlaB-PspA fusion protein were the most protected from a lethal challenge with live S. pneumoniae, as compared to the mice immunized with PspA only, a mixture of PspA and FlaB, or the PspA-FlaB fusion protein. FlaB-PspA also induced a cross protection against heterologous capsular types. These results suggest that a FlaB-PspA fusion protein alone could be used as an anti-pneumococcal mucosal vaccine or as an effective partner protein for multivalent capsular polysaccharide conjugate vaccines.     

Rectal immunization of mice with hepatitis A vaccine induces stronger systemic and local immune responses than parenteral immunization

Systemic (spleen cell (SPLC), serum antibodies) and intestinal mucosal (Peyer's patch cells (PPC), lamina propria lymphocytes (LPLs), coproantibodies) immune responses were compared in mice immunized with varying doses (144, 72, 36, 18 ELISA units [EU]) of HAVRIX, an alum-adsorbed killed hepatitis A virus (HAV) vaccine, delivered either intrarectally (i.r.) or intraperitoneally (i.p.) in three doses at weekly intervals. HAV-specific IgG, IgM, and IgA antibody responses were evaluated by ELISPOT and EIA and HAV-responsive lymphocytes by lymphocyte stimulation assays. Systemic IgG responses were greater in mice immunized intraperitoneally with 144, 72, and 36EU of HAVRIX, while IgM and IgA responses were greater in PPC and LPL cell populations, serum and coproantibodies of rectally immunized mice, particularly at HAVRIX doses of 36 and 18EU. Rectal immunization at lower doses (36, 18EU) also elicited strong cellular responses in all cell populations while parenteral (i.p.) vaccination, did not. Results suggest that rectal immunization may be a highly effective way of inducing both local and systemic immunity to HAV.     

Immunization with recombinant truncated Neisseria meningitidis-Macrophage Infectivity Potentiator (rT-Nm-MIP) protein induces murine antibodies that are cross-reactive and bactericidal for Neisseria gonorrhoeae

Neisseria meningitidis (Nm) and N. gonorrhoeae (Ng) express a Macrophage Infectivity Potentiator (MIP, NMB1567/NEIS1487) protein in their outer membrane (OM). In this study, we prepared independent batches of liposomes (n = 3) and liposomes + MonoPhosphoryl Lipid A (MPLA) (n = 3) containing recombinant truncated Nm-MIP protein encoded by Allele 2 (rT-Nm-MIP, amino acids 22–142), and used these to immunize mice. We tested the hypothesis that independent vaccine batches showed similar antigenicity, and that antisera could recognise both meningococcal and gonococcal MIP and induce cross-species bactericidal activity.The different batches of M2 rT-Nm-MIP-liposomes ± MPLA showed no significant (P > 0.05) batch-to-batch variation in antigenicity. Anti-rT-Nm-MIP sera reacted equally and specifically with Nm-MIP and Ng-MIP in OM and on live bacterial cell surfaces. Specificity was shown by no antiserum reactivity with Δmip bacteria. Using human complement/serum bactericidal assays, anti-M2 rT-Nm-MIP sera killed homologous meningococcal serogroup B (MenB) strains (median titres of 32–64 for anti-rT-Nm-MIP-liposome sera; 128–256 for anti-rT-Nm-MIP-liposome + MPLA sera) and heterologous M1 protein-expressing MenB strains (titres of 64 for anti rT-Nm-MIP-liposome sera; 128–256 for anti-rT-Nm-MIP-liposome + MPLA sera). Low-level killing (P < 0.05) was observed for a MenB isolate expressing M7 protein (titres 4–8), but MenB strains expressing M6 protein were not killed (titre < 4–8). Killing (P < 0.05) was observed against MenC and MenW bacteria expressing homologous M2 protein (titres of 8–16) but not against MenA or MenY bacteria (titres < 4–8).Antisera to M2 rT-Nm-MIP showed significant (P < 0.05) cross-bactericidal activity against gonococcal strain P9-17 (expressing M35 Ng-MIP, titres of 64–512) and strain 12CFX_T_003 (expressing M10 Ng-MIP, titres 8–16) but not against FA1090 (expressing M8 Ng-MIP).As an alternative to producing recombinant protein, we engineered successfully the Nm-OM to express M2 Truncated–Nm-MIP, but lipooligosaccharide-extraction with Na-DOC was contra-indicated. Our data suggest that a multi-component vaccine containing a select number of Nm- and Ng-MIP type proteins would be required to provide broad coverage of both pathogens.     

Intranasal immunization with recombinant antigens associated with new cationic particles induces strong mucosal as well as systemic antibody and CTL responses

New cationic nanoparticles (SMBV) were evaluated for use as a nasal vaccine delivery system for two recombinant proteins: HBsAg and beta-galactosidase. Each protein was formulated with SMBV and intranasally administrated to non-anesthetized mice. In each model, the formulated protein induced high levels of specific serum IgG antibodies and cytotoxic T lymphocyte (CTL) responses. Moreover, specific IgA antibodies were found in nasal as well as in vaginal washes of intranasally immunized mice with the protein associated with SMBV. In contrast, no IgG or IgA antibodies and no CTL were detected in mice immunized with free protein. The detection of a CTL response and an increase in both IgG1 and IgG2a antibodies in serum suggest that SMBV amplifies both Th1 and Th2 responses without modifying the Th1/Th2 profile of the immune response induced by the natural protein. These data demonstrate the high potential of SMBV for use as a nasal delivery system for sub-unit vaccines.     

A putative amino acid ABC transporter substrate-binding protein,NMB1612, from Neisseria meningitidis,induces murine bactericidal antibodies against meningococci expressing heterologous NMB1612 proteins

The nmb1612 (NEIS1533) gene encoding the ∼27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)₃ and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre <4) in a human serum bactericidal assay (hSBA) using anti-rNMB1612 sera, although another strain (MC168) expressing the same protein was killed (median titres of 16–64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015, >91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new meningococcal vaccines.     

Intranasal immunization with recombinant Vaccinia virus Tiantan harboring Zaire Ebola virus gp elicited systemic and mucosal neutralizing antibody in mice

Accumulating literature revealed that human mucosa was likely one of the important routes for EBOV attachment and further infection. Therefore inducing effective mucosal immune responses play key role in preventing the virus infection. Vaccinia virus Tiantan strain (VV) was a remarkably attenuated poxvirus, which has been broadly exploited as a multifunctional vector during the development of genetically recombinant vaccine and cancer therapeutic agent. In this study, we generated a recombinant VV harboring EBOV gp (VV-_Egp) that was used to immunize mice, followed by assessing immune responses, particularly the mucosal immune responses to EBOV GP. A stable and further attenuated VV-_Egp, in which the VV ha gene was replaced with the EBOV gp, was generated. In BALB/c mouse model, intranasal immunization with VV-_Egp elicited robust humoral and cellular immune responses, including high level of neutralizing serum IgG and IgA against EBOV, and a large amount of GP-specific IFN-γ secreting lymphocytes. More importantly, EBOV GP-specific neutralizing secreted IgA (sIgA) in nasal wash and both sIgA and IgG in vaginal wash were induced. In summary, immunization with a safe and stable recombinant VV carrying a single EBOV gp conferred robust systemic immune response and mucosal neutralizing antibodies, indicating that the recombinant virus could be utilized as a viral vector for plug-and-play universal platform in mucosal vaccine development.     

Development of an FHbp-CTB holotoxin-like chimera and the elicitation of bactericidal antibodies against serogroup B Neisseria meningitidis

The Neisseria meningitidis factor H binding protein (FHbp) is an important virulence factor and vaccine antigen contained in both USA licensed serogroup B meningococcal vaccines. Recent studies in human factor H (hFH) transgenic mice suggest that hFH-FHbp interactions lower FHbp-elicited immunogenicity. To provide tools with which to characterize and potentially improve FHbp immunogenicity, we developed an FHbp-cholera holotoxin-like chimera vaccine expression system in Escherichia coli that utilizes cholera toxin B (CTB) as both a scaffold and adjuvant for FHbp. We developed FHbp–CTB chimeras using a wild-type (WT) FHbp and a low hFH-binding FHbp mutant R41S. Both chimeras bound to G_M1 ganglioside and were recognized by the FHbp-specific monoclonal antibody JAR4. The R41S mutant had greatly reduced hFH binding compared to the WT FHbp-CTB chimera. WT and R41S FHbp-CTB chimeric antigens were compared to equimolar amounts of FHbp admixed with CTB or FHbp alone in mouse immunogenicity studies. The chimeras were significantly more immunogenic than FHbp alone or mixed with CTB, and elicited bactericidal antibodies against a panel of MenB isolates. This study demonstrates a unique and simple method for studying FHbp immunogenicity. The chimeric approach may facilitate studies of other protein-based antigens targeting pathogenic Neisseria and lay groundwork for the development of new protein based vaccines against meningococcal and gonococcal disease.     

Intranasal vaccination with a replication-deficient influenza virus induces heterosubtypic neutralising mucosal IgA antibodies in humans

We investigated the cross-neutralising potential of serum and nasal wash samples from volunteers who were intranasally immunised once with a monovalent replication-deficient delNS1-H1N1 influenza virus vaccine (7.7 log₁₀ TCID₅₀/volunteer). Eight out of twelve (8/12) vaccinees responded to vaccination with a significant increase of antibody levels in serum IgG ELISA, mucosal IgA ELISA, MNA or HAI. Four responders showed delNS1-specific ELISA IgA increases and revealed excellent homosubtypic neutralising activity in serum and mucosal washings (4/4). However, 0/4 of the sera but 3/4 of the nasal washings neutralised also heterosubtypic H3N2 and H5N1 influenza viruses. Depletion experiments proved that IgA but not IgG is responsible for the cross-neutralising activity of the nasal wash sample. Our findings indicate that the induction of virus-neutralising IgA may represent a valuable correlate of cross-protection of intranasal influenza vaccines and that the delNS1 concept constitutes a promising approach to protect humans from seasonal and pandemic influenza threats.     

Sublingual immunization with a subunit influenza vaccine elicits comparable systemic immune response as intramuscular immunization,but also induces local IgA and TH17 responses

Influenza is a vaccine-preventable disease that remains a major health problem world-wide. Needle and syringe are still the primary delivery devices, and injection of liquid vaccine into the muscle is still the primary route of immunization. Vaccines could be more convenient and effective if they were delivered by the mucosal route. Elicitation of systemic and mucosal innate and adaptive immune responses, such as pathogen neutralizing antibodies (including mucosal IgA at the site of pathogen entry) and CD4⁺ T-helper cells (especially the Th17 subset), have a critical role in vaccine-mediated protection. In the current study, a sublingual subunit influenza vaccine formulated with or without mucosal adjuvant was evaluated for systemic and mucosal immunogenicity and compared to intranasal and intramuscular vaccination. Sublingual administration of adjuvanted influenza vaccine elicited comparable antibody titers to those elicited by intramuscular immunization with conventional influenza vaccine. Furthermore, influenza-specific Th17 cells or neutralizing mucosal IgA were detected exclusively after mucosal immunization.     

Immunization of mice with recombinant gp41 in a systemic prime/mucosal boost protocol induces HIV-1-specific serum IgG and secretory IgA antibodies.

We tested the immunogenicity in mice of a recombinant fusion protein (gp41HA) consisting of the ectodomain of the HIV-1(IIIB) envelope glycoprotein gp41 fused to a fragment of the influenza virus HA2 hemagglutinin protein. An intraperitoneal prime followed by intranasal or intragastric boosts with gp41HA induced high concentrations of serum IgG antibodies and fecal IgA antibodies that reacted with gp41 in HIV-1(IIIB) viral lysate and were cross-reactive with gp41 in HIV-1(MN) lysate. By indirect immunofluorescence, serum IgG and fecal IgA from immunized mice were also shown to recognize gp41 in acetone-fixed human peripheral blood mononuclear cells infected with either syncytium-inducing (SI) or non-syncytium-inducing (NSI) North American HIV-1 field isolates, but not uninfected cells. Thus, this recombinant antigen may be useful in prime/boost immunization protocols designed to induce systemic and mucosal antibodies that recognize multiple primary HIV-1 isolates.     

Intranasal immunization of young mice with a multigene HIV-1 vaccine in combination with the N3 adjuvant induces mucosal and systemic immune responses

One of the major challenges for the development of an HIV vaccine is to induce potent virus-specific immune responses at the mucosal surfaces where transmission of virus occurs. Intranasal delivery of classical vaccines has been shown to induce good mucosal antibody responses, but so far for genetic vaccines the success has been limited. This study shows that young individuals are sensitive to nasal immunization with a genetic vaccine delivered in a formulation of a lipid adjuvant, the Eurocine N3. Intranasal delivery of a multiclade/multigene HIV-1 genetic vaccine gave rise to vaginal and rectal IgA responses as well as systemic humoral and cellular responses. As electroporation might become the preferred means of delivering genetic vaccines for systemic HIV immunity, nasal delivery by droplet formulation in a lipid adjuvant might become a means of priming or boosting the mucosal immunity.     

Intranasal immunization with recombinant Vaccinia virus encoding trimeric SARS-CoV-2 spike receptor-binding domain induces neutralizing antibody

Respiratory transmission of SARS-CoV-2 is considered to be the major dissemination route for COVID-19, therefore, mucosal immune responses have great importance in preventing SARS-CoV-2 from infection. In this study, we constructed a recombinant Vaccinia virus (VV) harboring trimeric receptor-binding domain (RBD) of SARS-CoV-2 spike protein (VV_-tRBD), and evaluated the immune responses towards RBD following intranasal immunization against mice and rabbits. In BALB/c mice, intranasal immunization with VV_-tRBD elicited robust humoral and cellular immune responses, with high-level of both neutralizing IgG and IgA in sera against SARS-CoV-2 psudoviruses, and a number of RBD-specific IFN-γ-secreting lymphocytes. Sera from immunized rabbits also exhibited neutralization effects. Notably, RBD-specific secretory IgA (sIgA) in both nasal washes and bronchoalveolar lavage fluids (BALs) were detectable and showed substantial neutralization activities. Collectively, a recombinant VV expressing trimeric RBD confers robust systemic immune response and mucosal neutralizing antibodies, thus warranting further exploration as a mucosal vaccine.     

Intranasal immunization with a mixture of PspA and a Toll-like receptor agonist induces specific antibodies and enhances bacterial clearance in the airways of mice

To develop an effective nasal vaccine for Streptococcus pneumoniae, the effects of a panel of Toll-like receptor (TLR) agonists in combination with pneumococcal surface protein A (PspA) on induction of PspA-specific antibodies and bacterial clearance were compared in mice. Mice were nasally immunized with 10 μg of TLR agonist (TLR 2–4 and 9) and 2.5 μg of PspA once per week for 3 weeks. Significantly increased levels of PspA-specific immunoglobulin G (IgG) and IgA in the airways and PspA-specific IgG in plasma were found in mice administered PspA plus each TLR agonist, compared with mice administered PspA alone. In a sub-lethal pneumonia model using a serotype 3 pneumococcal strain, bacterial density in the lungs of mice was significantly reduced in mice administered PspA plus each TLR agonist, compared with mice administered either PspA alone or phosphate-buffered saline alone 3 h after bacterial challenge. Similarly, enhanced bacterial clearance was found in the nasopharynx of mice administered PspA plus each TLR agonist 1 day after infection with a serotype 19F strain. Our data suggest that PspA-specific antibody induced by nasal immunization with PspA plus TLR agonist is capable of reducing the bacterial load in both the nasopharynx and lungs after challenge with pneumococci with different serotypes. Despite the skewed Th1/Th2 immune responses, the effects of nasal immunization with PspA plus each TLR agonist on bacterial clearances from the lungs 3 h after infection and from nasopharynx 1 day after infection in mice were equivalent.     

Sublingual immunization induces broad-based systemic and mucosal immune responses in mice

The potential of sublingual (s.l.) delivery of vaccine was examined in mice. We show the existence of a dense network of dendritic cells (DCs) in the s.l. epithelium and a rapid and transient increase in the frequency of s.l. DCs after topical application of cholera toxin (CT) adjuvant under the tongue. S.l. immunization with ovalbumin and CT induced vigorous systemic and mucosal antibody responses. Such treatment promoted mixed Th1 and Th2 cytokine responses and induced cytotoxic CD8(+) T cells in lung tissues and in systemic lymphoid organs. S.l. immunization was comparable to intranasal immunization and was superior to oral immunization regarding the magnitude and anatomic dissemination of the induced immune responses. S.l. administration of live influenza virus at a dose lethal by the nasal route was well tolerated and did not redirect virus to the olfactory bulb. These features underscore the potential of the s.l. mucosa to serve as an alternative vaccine delivery route.