Humoral immunogenicity of an HIV-1 envelope residue 649-684 membrane-proximal region peptide fused to the plague antigen F1-V

The membrane-proximal region spanning residues 649-684 of the HIV-1 envelope protein gp41 (MPR_649-684) is an attractive vaccine target for humoral immunity that blocks viral transcytosis across the mucosal epithelia. However, induction of high-titer MPR_649-684-specific antibodies remains a challenging task. To explore potential solutions for this challenge, we tested a new translational fusion protein comprising the plague F1-V antigen and MPR_649-684 (F1-V-MPR_649-684). We employed systemic immunization for initial feasibility analyses. Despite strong immunogenicity demonstrated for the immunogen, repeated systemic immunizations of mice with F1-V-MPR_649-684 hardly induced MPR_649-684-specific IgG. In contrast, a single immunization with F1-V-MPR_649-684 mounted a significant anti-MPR_649-684 IgG response in animals that were primed with another MPR_649-684 fusion protein based on the cholera toxin B subunit. Additional boost immunizations with F1-V-MPR_649-684 recalled and maintained the antibody response and expanded the number of specific antibody-secreting B cells. Thus, while F1-V-MPR_649-684 alone was not sufficiently immunogenic to induce detectable levels of MPR_649-684-specific antibodies, these results suggest that prime-boost immunization using heterologous antigen-display platforms may overcome the poor humoral immunogenicity of MPR_649-684 for the induction of durable humoral immunity. Further studies are warranted to evaluate the feasibility of this strategy in mucosal immunization. Lastly, our findings add to a growing body of evidence in support of this strategy for immunogen design for poorly immunogenic epitopes besides the MPR of HIV-1's transmembrane envelope protein.     

Effect of pre-existing immunity for systemic and mucosal immune responses to intranasal immunization with group B Streptococcus type III capsular polysaccharide-cholera toxin B subunit conjugate

The effects of priming with a group B Streptococcus type III capsular polysaccharide (GBS CPS III)-recombinant cholera toxin B subunit (rCTB) conjugate, purified GBS CPS III or rCTB alone on the systemic and mucosal immune responses to CPS III after intranasal (i.n.) immunization were investigated in mice. Priming with purified GBS CPS III followed by boosting with GBS CPS III-rCTB conjugate or priming with the conjugate followed by boosting with free CPS induced comparable levels of specific IgG and IgA in both serum and in lungs and vagina. However, i.n. immunization comprising both priming and boosting with conjugate was superior to priming with CPS and boosting with conjugate or the reverse, especially with regard to inducing mucosal IgA anti-CPS responses. All the immunization schemes, except priming and boosting with free CPS, induced high and similar levels of IgG1 in serum. In contrast, mice primed with free CPS III and then boosted with CPS III-rCTB conjugate by the i.n. route failed to produce significant levels of IgG2a, IgG2b and IgG3 in serum, at difference from mice primed with the conjugate and boosted with either conjugate or free CPS. Pre-immunization with rCTB either i.n. or i.p. did not suppress specific serum IgG responses induced by GBS CPS III-rCTB conjugate intranasally, but did inhibit serum and especially mucosal IgA responses. Our findings suggest that priming with CPS affects the distribution of IgG subclasses to GBS CPS and that pre-existing anti-carrier rCTB immunity can have an inhibitory effect on mucosal immune responses elicited by the conjugate vaccine given by the i.n. route.     

Comparison of mucosal and systemic humoral immune responses after transcutaneous and oral immunization strategies

In order to compare the ability of transcutaneous and oral immunization strategies to induce mucosal and systemic immune responses, we inoculated mice transcutaneously with cholera toxin (CT) or the non-toxic B subunit of cholera toxin (CtxB), or orally with Peru2(pETR1), an attenuated vaccine strain of Vibrio cholerae expressing CtxB. In addition, we also evaluated dual immunization regimens (oral inoculation with transcutaneous boosting, and transcutaneous immunization with oral boosting) in an attempt to optimize induction of both mucosal and systemic immune responses. We found that transcutaneous immunization with purified CtxB or CT induces much more prominent systemic IgG anti-CtxB responses than does oral inoculation with a vaccine vector strain of V. cholerae expressing CtxB. In comparison, anti-CtxB IgA in serum, stool and bile were comparable in mice either transcutaneously or orally immunized. Overall, the most prominent systemic and mucosal anti-CtxB responses occurred in mice that were orally primed with Peru2(pETR1) and transcutaneously boosted with CT. Our results suggest that combination oral and transcutaneous immunization strategies may most prominently induce both mucosal and systemic humoral responses.     

Induction of HIV-1 MPR(649-684)-specific IgA and IgG antibodies in caprine colostrum using a peptide-based vaccine

Induction of antigen-specific antibodies against HIV-1 in colostrum and milk may help prevent breast milk transmission of the virus. A peptide vaccine against the HIV-1 gp41 membrane proximal region (MPR(649-684)) was evaluated as proof-of-principle in a caprine model. Pregnant Alpine/Saanen goats were immunized with MPR(649-684) peptide conjugated to KLH using alum adjuvant. Immunizations were intramuscular, intranasal, and in the supramammary lymph node region. Samples collected after parturition demonstrated the presence of MPR(649-684)-specific antibodies in colostrum and serum. These results support the concept that a peptide vaccine can effectively induce MPR(649-684)-specific sIgA and IgG in the colostrum of a lactating species.     

Mucosal priming with replicative Tiantan vaccinia and systemic boosting with DNA vaccine raised strong mucosal and systemic HIV-specific immune responses

An effective vaccine strategy for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity at mucosal and systemic sites. We tested a new prime-boost approach of intranasal priming with 3 × 10⁶ PFU of replicative recombinant Tiantan vaccinia virus (rTTV) and intramuscular boosting with 100 μg DNA plasmid expressing HIV-1 Gag in BALB/c mice along with other strategies. Our data demonstrated that intranasal priming with replicative recombinant Tiantan vaccinia and intramuscular boosting with DNA vaccine raised the highest vaginal IgA and systemic T-cell responses, and modest lung IgA and sera IgG responses among all vaccination regimens; each vaccination regimen generated its own imprint of the most preferential T-cell receptor usage of Vβ. These results demonstrate that the combination of intranasal priming with replicative recombinant Tiantan vaccinia and intramuscular boosting with DNA vaccine is a preferable regimen for induction of both T-cell and humoral immune responses at mucosal and systemic sites.     

A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM(197)) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 responses following nasal delivery

The development of new generation vaccines against diphtheria is dependent on the identification of antigens and routes of immunization that are capable of stimulating immune responses similar to, or greater than, those obtained with the parenterally-delivered toxoid vaccine, while reducing the adverse effects that have been associated with the traditional vaccine. In this study, we examined the cellular and humoral immune responses in mice generated after both parenteral and mucosal immunizations with cross-reacting material (CRM(197)) of diphtheria toxin. We found that both native and mildly formaldehyde-treated CRM(197) and conventional diphtheria toxoid (DT) induced mixed Th1/Th2 responses and similar levels of anti-DT serum IgG following parenteral immunization. In contrast, CRM(197) preparations were poorly immunogenic when administered intranasally in solution. However, formulation of the antigens with chitosan significantly enhanced their immunogenicity, inducing high levels of antigen-specific IgG, secretory IgA, toxin-neutralizing antibodies and T cell responses, predominately of Th2 subtype. Furthermore, intranasal immunization with CRM(197) and chitosan induced protective antibodies against the toxin in a guinea pig passive challenge model. We also found that priming parenterally with DT in alum and boosting intranasally with CRM(197) was a very effective method of immunization in mice, capable of inducing high levels of anti-DT IgG and neutralizing antibodies in the serum and secretory IgA in the respiratory tract. Our findings suggest that boosting intranasally with CRM(197) antigen may be very effective in adolescents or adults who have previously been parenterally immunized with a conventional diphtheria toxoid vaccine.     

The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant

Systemic and mucosal immune responses were effectively induced following the subcutaneous administration of Haemophilus influenzae type b oligosaccharide conjugated to diphtheria toxoid vaccine in a formulation containing the active form of vitamin D3. IgA and IgG antibodies with specificity for both the protein and oligosaccharide components of the vaccine were detectable in mucosal secretions following immunization. The IgA and IgG mucosal antibodies were produced locally, and were functional as demonstrated by their diphtheria toxin neutralizing activity. Our data suggests that subcutaneous tissues can effectively serve as effective antigen presenting sites for both mucosal and systemic immune responses to antigens administered in combination with vitamin D3.     

Frequent nasal administrations of recombinant cholera toxin B subunit (rCTB)-containing tetanus and diphtheria toxoid vaccines induced antigen-specific serum and mucosal immune responses in the presence of anti-rCTB antibodies

Vaccination via a mucosal route is a very attractive means for immunization, because both local and systemic immune responses are inducible and vaccines can be administered easily and safely from infants to elderly persons. For developing widely applicable mucosal vaccines using recombinant cholera toxin B subunit (rCTB) as a safe adjuvant, we examined whether frequent nasal administrations of rCTB-containing same and different vaccines could induce antigen-specific immune responses without induction of systemic tolerance and suppression by pre-existing anti-rCTB immunity. Ten repetitive nasal administrations to mice of tetanus toxoid (TT) + rCTB or diphtheria toxoid (DT) + rCTB raised and maintained high levels of antigen- and rCTB-specific serum IgG including high levels of tetanus/diphtheria antitoxin titres and raised nasal, salivary, lung, vaginal and fecal secreted IgA, suggesting that the regimen did not induce systemic tolerance to TT/DT and rCTB. Mice successively received repetitive five doses of TT as the first antigen and subsequent five doses of DT as the second antigen, and vice versa, raised serum IgG to the second antigen at various levels including low but sufficient protective levels of antitoxin titres and induced mucosal IgA in the lungs, the vaginas and feces, but hardly in the nasal secretions and salivas. After an interval of 22 weeks between the dosage of the first and second antigens, mice induced serum IgG to the second antigen at high levels and mucosal IgA in all sites. In conclusion, anti-TT and -DT serum and mucosal antibody responses induced by repeated intranasal immunization using rCTB adjuvant lasted for a long period, and for improving the effectivity of vaccination, different rCTB-containing vaccines should be administered at appropriate intervals.     

Prime-boost vaccine regimen confers protective immunity to human-derived enterotoxigenic Escherichia coli

Development of effective vaccines against diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains is still a priority for those living at or traveling to endemic regions. In this work, we evaluated the protective role of an anti-ETEC vaccine regimen based on parenteral priming with a DNA vaccine, pRECFA, followed by oral boosting with a recombinant attenuated Salmonella Typhimurium vaccine strain, HG3, both encoding the same antigen, the structural subunit (CfaB) of the ETEC CFA/I fimbriae. The DNA-priming Salmonella-boosting protocol enhanced both murine anti-CfaB serum IgG and fecal IgA antibody responses and increased the ability of serum antibodies to inhibit the adhesive properties of the CFA/I fimbriae expressed by live bacteria, as compared to mice immunized with only one vaccine type. Addition of a mucosal adjuvant (LTR192G) to the Salmonella vaccine strain further enhanced the synergic effects of the vaccine regimen on the induced CfaB-specific antibody responses. DBA/2 dams submitted to the prime-boost regimen transferred complete passive protection to suckling neonates challenged with a virulent ETEC strain. Detection of milk anti-CfaB IgA antibodies and protection conferred by vaccinated dams to neonates born from non-vaccinated dams indicated that secretion of antigen-specific IgA is the immune response induced by the protective vaccine regimen. These results demonstrate that priming with a DNA vaccine and boosting with a Salmonella strain enhances both quantitatively and qualitatively the antibody responses to the CfaB antigen and represents an alternative for either active or passive immunization approach to ETEC-associated diarrhea.     

Enhanced mucosal and systemic immune responses to a vaginal vaccine coadministered with RANTES-expressing plasmid DNA using in situ-gelling mucoadhesive delivery system

To develop more potent and convenient mucosal vaccines, we investigated the effect of an in situ-gelling mucoadhesive vaginal vaccine delivery system and a genetic chemokine adjuvant on the local and systemic immune responses. The in situ-gelling mucoadhesive delivery system of hepatitis B surface antigen (HBsAg), composed of poloxamers and polycarbophil, showed the prolonged retention at the vaginal tissues. Following intravaginal administration to mice, HBsAg-specific IgA was induced in the vagina and saliva, and IgG was produced in the serum. RANTES-expressing plasmid (pRANTES) intravaginally coadministered with HBsAg showed the expression at the vaginal tissues, and more effectively induced the vaginal IgA and serum IgG immune responses than did cholera toxin (CT). The intramuscular coadministration of pRANTES with HBsAg also increased both serum IgG levels and mucosal IgA levels. Regardless of the adjuvants, the in situ-gelling mucoadhesive HBsAg delivery system enhanced the mucosal and systemic immune responses. At 42 days after the first immunization, the highest vaginal IgA levels were induced after intravaginal immunization of HBsAg plus pRANTES using the in situ-gelling mucoadhesive delivery system, showing 182- and 1035-fold higher titer compared to the groups receiving HBsAg alone in PBS by intravaginal and intramuscular routes, respectively. Our results indicate that the use of in situ-gelling mucoadhesive delivery systems with the genetic chemokine adjuvant pRANTES would be advantageous for more effective induction of mucosal and systemic immune responses to intravaginally administered vaccines.     

Nasal immunization with E. coli verotoxin 1 (VT1)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies

Escherichia coli O157:H7 produces two forms of verotoxin (VT), VT1 and VT2, which cause hemorrhagic colitis with development, in some cases, of hemolytic uremic syndrome. These toxins consist of an enzymatically active A subunit and pentamers of B subunit responsible for their binding to host cells. We used the secretion-expression system of Bacillus brevis to produce recombinant VT1B and VT2B. The secreted B subunits were purified and sequenced to verify their structure. Receptor-binding showed that rVT1B but not rVT2B bound to Gb3-receptor. When mice were nasally immunized with rVT1B or rVT2B together with a nontoxic mutant of cholera toxin (mCT) or native cholera toxin (nCT) as adjuvants, serum IgG and mucosal IgA antibody responses to VT1B were induced. The VT1B-specific antibodies prevented VT1B binding to its Gb3 receptor. In contrast, poor serum and no mucosal VT2B-specific antibodies but brisk CTB-specific antibody responses were induced by nasal immunization with rVT2B in the presence of mCT or nCT. These results show that nasal immunization with rVTB and mCT as a nontoxic mucosal adjuvant is an effective regimen for the induction of VT1B but not VT2B antibody responses which inhibit VT1B binding to Gb3 receptor.     

Role of CD80 and CD86 in host immune responses to the recombinant hemagglutinin domain of Porphyromonas gingivalis gingipain and in the adjuvanticity of cholera toxin B and monophosphoryl lipid A

The gingipains of Porphyromonas gingivalis have been implicated in the virulence of this bacterium, and antibodies to the hemagglutinin/adhesin domain (HArep) of the gingipains have been shown to protect against P. gingivalis colonization. However, the cellular mechanisms involved in host responses to HArep have not been elucidated. The purpose of the present study was to determine the functional role of CD80 and CD86 in mediating systemic and mucosal immune responses to the recombinant HArep derived from the gingipain Kgp (Kgp-HArep) after intranasal (i.n.) immunization. We also investigated the effect of the mucosal adjuvants the B subunit of cholera toxin (CTB) and monophosphoryl lipid A (MPL) on the functional role of the costimulatory molecules for the induction of systemic and mucosal responses to Kgp-HArep. The in vivo functional roles of CD80 and CD86 were assessed in C57BL/6 wild-type (wt), CD80(-/-), CD86(-/-) and CD80/CD86(-/-) mice following intranasal immunization with Kgp-HArep with or without adjuvant. Serum IgG and mucosal IgA antibody responses were induced following i.n. immunization of mice with Kgp-HArep, and were potentiated by CTB or MPL. A differential requirement of CD80and/or CD86 was observed for systemic IgG anti-Kgp-HArep responses following the primary and secondary immunization with antigen alone or antigen+adjuvant. Compared to wt and CD80(-/-) mice, CD86(-/-) mice had reduced serum IgG anti-Kgp-HArep responses following the second immunization with antigen alone or antigen+CTB, whereas similar levels of serum IgG anti-Kgp-HArep antibody activity were observed in wt, CD80(-/-) and CD86(-/-) mice immunized with antigen+MPL. Analysis of the serum IgG subclass responses revealed that CD80 influenced both Th1- and Th2-like IgG subclass responses, while CD86 preferentially influenced a Th2-associated IgG subclass response to Kgp-HArep. Mucosal IgA anti-Kgp-HArep responses in saliva and vaginal washes were diminished in CD86(-/-) mice. In vitro stimulation of murine bone marrow-derived dendritic cells with Kgp-HArep, CTB and MPL resulted in an up-regulation of CD80 and especially CD86 expression. Taken together, our results demonstrate that CD80 and CD86 can play distinct as well as redundant roles in mediating a systemic immune response and that CD86 plays a unique role in mediating a mucosal response to Kgp-HArep following immunization via the i.n. route alone or with adjuvant.     

Oral immunization of rhesus macaques with adenoviral HIV vaccines using enteric-coated capsules

Targeted delivery of vaccine candidates to the gastrointestinal (GI) tract holds potential for mucosal immunization, particularly against mucosal pathogens like the human immunodeficiency virus (HIV). Among the different strategies for achieving targeted release in the GI tract, namely the small intestine, pH sensitive enteric coating polymers have been shown to protect solid oral dosage forms from the harsh digestive environment of the stomach and dissolve relatively rapidly in the small intestine by taking advantage of the luminal pH gradient. We developed an enteric polymethacrylate formulation for coating hydroxy-propyl-methyl-cellulose (HPMC) capsules containing lyophilized Adenoviral type 5 (Ad5) vectors expressing HIV-1 gag and a string of six highly-conserved HIV-1 envelope peptides representing broadly cross-reactive CD4⁺ and CD8⁺ T cell epitopes. Oral immunization of rhesus macaques with these capsules primed antigen-specific mucosal and systemic immune responses and subsequent intranasal delivery of the envelope peptide cocktail using a mutant cholera toxin adjuvant boosted cellular immune responses including, antigen-specific intracellular IFN-γ-producing CD4⁺ and CD8⁺ effector memory T cells in the intestine. These results suggest that the combination of oral adenoviral vector priming followed by intranasal protein/peptide boosting may be an effective mucosal HIV vaccination strategy for targeting viral antigens to the GI tract and priming systemic and mucosal immunity.     

Induction of systemic and mucosal antibody responses in mice immunized intranasally with aluminium-non-adsorbed diphtheria toxoid together with recombinant cholera toxin B subunit as an adjuvant

Nasal mucosal immunization is very attractive for vaccination to prevent various bacterial and viral infectious diseases because of induction of systemic and mucosal immune responses. The aim of the present study was to investigate the possibility of changing the immunization procedure of diphtheria toxoid (Dt) from intramuscular or subcutaneous injection to intranasal administration. Intranasal immunization with aluminium-non-adsorbed diphtheria toxoid (nDt) together with recombinant cholera toxin B subunit (rCTB, 10 μg) induced, at a concentration of 5 Lf, high levels of serum Dt-specific IgG antibody responses and high or moderate levels of the specific IgA antibody responses in all mice and only a slight level of the specific IgE antibody responses in some mice. Furthermore, sufficiently high diphtheria antitoxin titres more than 0.1 international units (IU) ml⁻¹ were obtained from mice which showed high levels of serum Dt-specific IgG antibody responses. Under the same experimental conditions, induction of significant levels of mucosal Dt-specific IgA antibody responses occurred in the nasal cavity, the lung, the saliva and vaginal secretions and the small and large intestines of all mice, although there were different titres between individual mice. Similar results were also obtained with rCTB-specific serum IgG and IgA and mucosal IgA antibody responses; serum rCTB-specific IgE antibody titres were not detected. These results show that intranasal administration of nDt with rCTB must be a very useful means for vaccination against diphtheria.     

Enhancement of mucosal immune response against HIV-1 Gag by DNA immunization

In order to examine the feasibility of Gag-expression DNA as a potential candidate for HIV vaccine using a mouse model, we injected DNA into mice either intramuscularly or by using a gene gun. Both methods induced a low level of antibody production. However, after booster immunization with p24 protein emulsified with complete Freund's adjuvant via a footpad, we found that only the preceding intramuscular DNA immunization induced an anti-Gag Th1-type (IgG(2a)) antibody response, in addition to the enhancement of a Th2-type (IgG(1)) antibody response. Importantly, when mice were boosted intranasally with p24 and cholera toxin, intramuscular DNA injection was found to enhance both systemic and mucosal Gag-specific immune responses. These results indicate that intramuscular DNA immunization confers the inducibility of memory cells, which circulate around various mucosal tissues. Therefore, intramuscular DNA priming, followed by a mucosal booster immunization, could be considered as a regimen applicable to HIV vaccine.     

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.     

Mucosal priming with PEI/DNA complex and systemic boosting with recombinant TianTan vaccinia stimulate vigorous mucosal and systemic immune responses

An effective vaccine strategy for HIV-1 will probably requires the induction and maintenance of both humoral and cellular immunity. We tested a new prime-boost approach of intranasal priming with 10 microg DNA plasmid in the PEI/DNA complexes and boosting with 10(7)PFU of replicative recombinant TianTan vaccinia virus (rTTV) expressing HIV-1 Gag in BALB/c mice. Intranasal priming with PEI/DNA complexes elicited strikingly stronger HIV-specific T-cell (p=0.0358) and IgA immune responses at mucosal sites of lung (p=0.0445) and vaginal tract (p=0.0469) than intranasal priming with naked DNA, though both are followed by the same rTTV boosting. Furthermore, an intramuscular boosting with rTTV could profoundly enhance both T-cell and antibody immune responses raised by intranasal priming. These results demonstrate that the combination of intranasal priming with PEI/DNA complexes and systemic boosting with rTTV is a preferable regimen for induction of both T-cell and humoral immune responses.     

Mucosal administration of a chimera composed of Pseudomonas exotoxin and the gp120 V3 loop sequence of HIV-1 induces both salivary and serum antibody responses

We have used a mouse immunization model to evaluate the potential for a chimera protein composed of a nontoxic form of Pseudomonas exotoxin (ntPE) to incite and sustain a mucosal immune response against an integrated antigen. The chimera, termed ntPE-V3MN26, contained 26 amino acids of the gp120 V3 loop region sequence of the MN strain of HIV-1 integrated in place of the Ib region of ntPE. Following either vaginal, rectal, oral or subcutaneous administration and boosting, anti-gp120-specific IgA and IgG levels in serum and saliva samples were assessed by ELISA. All dosing regimens stimulated significant and comparable salivary IgA and serum IgG responses at 1, 2 and 3 months after the initial inoculation. Following a boost at 16 months with ntPE-V3MN26, a strong memory response to the antigen was observed. Isotyping of serum antibodies at this time suggested that both a Thl and a Th2 response had been induced. Responses to ntPE-V3MN26 following subcutaneous injection in the presence or absence of Freund's adjuvant demonstrated that Freund's adjuvant resulted in a three-fold greater enhancement of immune response compared to administration of chimera alone. These results demonstrate that mucosal presentation of a chimera composed of a nontoxic form of Pseudomonas exotoxin can result in a strong mucosal and systemic antigen-specific immune response to an integrated antigen. The profound memory responses induced by this chimera may be particularly useful for practical vaccine applications.     

Preparation and preclinical evaluation of experimental group B streptococcus type III polysaccharide-cholera toxin B subunit conjugate vaccine for intranasal immunization

Streptococcus group B (GBS) is usually carried asymptomatically in the vaginal tract of women and can be transferred to the newborn during parturition. Serum antibodies to the capsular polysaccharide (CPS) can prevent invasive diseases, whereas immunity acting at the mucosal surface may be more important to inhibit the mucosal colonization of GBS and thus the risk of infection for the newborn. We prepared different GBS type III CPS-protein conjugate vaccines and evaluated their systemic and mucosal immunogenicity in mice. GBS type III CPS was conjugated to tetanus toxoid (TT) or recombinant cholera toxin B subunit (rCTB) either directly or to rCTB indirectly via TT. The conjugation was performed by different methods: (1) CPS was coupled to TT with 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide (EDAC), using adipic acid dihydrazide (ADH) as a spacer; (2) CPS was conjugated with rCTB using reductive amination; or, (3) N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) was used to bind rCTB to the TT of the CPS-TT conjugate. Mice were immunized with these conjugates or purified CPS by subcutaneous (s.c.) and intranasal (i. n.) routes. Antibodies to GBS III in serum, lungs and vagina were measured with ELISA. All of the CPS-protein conjugates were superior to unconjugated CPS in eliciting CPS-specific immune responses in serum and mucosal tissue extracts. The conjugates, when administrated s.c., induced only IgG responses in serum, lung and vagina, while i.n. vaccination also elicited IgA responses in the lungs and vagina. The CPS-TT conjugate administrated i.n. induced a strong serum IgG, but only a weak mucosal IgA response, while the CPS-rCTB conjugate elicited high IgG as well as IgA antibodies in the lungs after i.n. immunization. GBS III CPS-TT conjugated with rCTB produced a strong systemic and local anti-CPSIII response after i.n. administration. Co-administration of CT as adjuvant enhanced the anti-CPS systemic and mucosal immune responses further after i.n. administration with the CPS conjugates. These findings indicate that: (i) i.n. immunization with GBS CPS-protein conjugates was more effective than s.c immunization for stimulating serum as well as mucosal immune responses; (ii) rCTB as a carrier protein for GBS III CPS could markedly improve the mucosal immune response; and (iii) the experimental GBS type III CPS conjugates containing rCTB should be investigated as mucosal vaccine to prevent GBS infection in humans.     

Intranasal immunization with outer membrane protein P6 and cholera toxin induces specific sinus mucosal immunity and enhances sinus clearance of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) is one of the leading pathogens in sinusitis. One of the outer membrane proteins of NTHi, P6, is a common antigen to all strains and is an attractive candidate for a subunit bacterial vaccine. In this study, we characterized normal sinus mucosa (SM) and investigated the potential of intranasal immunization with P6 and cholera toxin (CT) for induction of mucosal protective immunity against NTHi in the maxillary sinuses of rats. Intranasal immunization induced P6-specific sinus mucosal and systemic immunological responses, mainly of the IgA and IgG isotype. The protective effect of intranasal immunization was demonstrated by enhancement of sinus clearance of NTHi. The present study showed that unilateral intranasal immunization has a capacity to induce protective immunity against NTHi in the bilateral maxillary sinuses. Systemic administration of the vaccine did not affect sinus clearance of NTHi. These findings suggest that a nasal vaccine might be useful for preventing sinusitis.