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.     

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.     

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.     

Mucosal and systemic antibody responses against an acellular pertussis vaccine in mice after intranasal co-administration with recombinant cholera toxin B subunit as an adjuvant

To investigate the possibility of intranasal immunization with an acellular pertussis vaccine, groups of mice were administered intranasally with aluminium-non-adsorbed pertussis toxoid (PTd; 0.5 or 5 microg) and formalin-treated filamentous hemagglutinin (fFHA; 5 microg) with and without recombinant cholera toxin B subunit (rCTB; 10 microg) as a mucosal adjuvant. At a low concentration of PTd, the following things became clear: (1) earlier and higher elevation of serum anti-PTd and anti-FHA IgG antibody titres in the presence of rCTB than in its absence, (2) higher serum anti-PTd and anti-FHA IgG antibody titres than 200 and 100 ELISA units ml(-1) (EU ml(-1)) in all mice, respectively, in the presence of rCTB, which were obtained by calibration against a reference anti-pertussis mouse serum, and (3) in an intranasal challenge experiment with Bordetella pertussis, slightly more rapid elimination of the bacteria from the lungs of mice intranasally immunized in the presence of rCTB, suggesting the effectiveness of rCTB as a mucosal adjuvant. However, irrespective of rCTB and dose of PTd, mice which were immunized four times and sacrificed on day 35 developed high levels of anti-PTd serum IgG antibodies, high or moderate levels of anti-FHA serum IgG antibodies and mucosal anti-PTd IgA antibodies in the lungs; only a slight or no increase of anti-FHA mucosal IgA antibodies was observed in the lung. These facts suggested the immunogenicity and mucosal adjuvanticity of PTd, and therefore, the mucosal adjuvanticity of rCTB seemed to be inconspicuous. Moreover, the addition of rCTB induced higher anti-PTd serum IgE antibody responses than no addition of it depending on dose of PTd. These results show that dose of PTd included in an acellular pertussis vaccine had better be low as possible and the addition of rCTB may not be always necessary in case of this nasal vaccine alone unlike tetanus and diphtheria toxoids and hepatitis B virus vaccine reported before.     

Outer membrane vesicles of Neisseria lactamica as a potential mucosal adjuvant

The muscosal delivery of vaccines has many advantages including ease of administration and the induction of a mucosal immune response at the natural site of infection for many pathogens. Mice were immunised with outer membrane vesicles (OMV) prepared from Neisseria lactamica or Neisseria meningitidis by subcutaneous (SC) or intranasal (IN) routes, or live cells of N. lactamica given IN or by SC injection. A systemic IgG and mucosal IgA response was demonstrated and N. lactamica OMV induced antibodies cross-reactive with N. meningitidis; however, a cross-reactive response following IN administration was only evident after three doses of vaccine. OMV from both organisms were also an effective intranasal adjuvant for a co-administered model antigen, hepatitis B surface antigen (HBsAg), inducing systemic IgG against HBsAg and IgA in lung and vaginal washes. IN administration of N. meningitidis OMV elicited serum antibodies that were bactericidal for meningococci and provided passive protection in an infant rat model of meningococcal bacteraemia. The antibody response to N. lactamica OMV given IN was only weakly bactericidal but still afforded passive protection. Thus, OMV from N. lactamica given IN elicit immune responses cross-reactive with N. meningitidis and act as an effective mucosal adjuvant.     

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.     

Recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant enhances induction of diphtheria and tetanus antitoxin antibodies in mice by intranasal administration with diphtheria-pertussis-tetanus (DPT) combination vaccine

Recombinant cholera toxin B subunit (rCTB) which is produced by Bacillus brevis carrying pNU212-CTB acts as a mucosal adjuvant capable of enhancing host immune responses specific to unrelated, mucosally co-administered vaccine antigens. When mice were administered intranasally with diphtheria-pertussis-tetanus (DPT) combination vaccine consisting of diphtheria toxoid (DTd), tetanus toxoid (TTd), pertussis toxoid (PTd), and formalin-treated filamentous hemagglutinin (fFHA), the presence of rCTB elevated constantly high values of DTd- and TTd-specific serum ELISA IgG antibody titres, and protective levels of diphtheria and tetanus toxin-neutralizing antibodies but the absence of rCTB did not. Moreover, the addition of rCTB protected all mice against tetanic symptoms and deaths. DPT combination vaccine raised high levels of serum anti-PT IgG antibody titres regardless of rCTB and protected mice from Bordetella pertussis challenge. These results suggest that co-administration of rCTB as an adjuvant is necessary for induction of diphtheria and tetanus antitoxin antibodies on the occasion of intranasal administration of DPT combination vaccine.     

Intranasal immunisation with conjugate vaccine protects mice from systemic and respiratory tract infection with Pseudomonas aeruginosa

We tested intranasal application of anti-Pseudomonas conjugate vaccine in mice. Comparison of immunisation via the intra-muscular versus intranasal routes showed the induction of equivalent levels of specific serum IgG and IgG subclasses antibodies if cholera toxin was used as an adjuvant. In contrast, secretion of specific mucosal IgA antibodies in the upper respiratory tract was only observed after intranasal immunisation together with adjuvant. Systemic and mucosal immunity was also established via the intranasal route when CpG-containing oligonucleotides were used as adjuvant. The functionality of intranasally induced antibodies was proven in vitro by opsonophagocytosis and in vivo using the burn-wound sepsis and intra-tracheal lung infection models. These results demonstrate the feasibility of intranasal immunisation against P. aeruginosa with conjugate vaccine.     

Highly purified mutant E112K of cholera toxin elicits protective lung mucosal immunity to diphtheria toxin.

We demonstrated that the mutant of cholera toxin (mCT) E112K which was LPS-free supported the induction of protective immunity in mucosal (e.g. lung lavage) and systemic (e.g. serum) compartments when given nasally with vaccine-grade diphtheria toxoid (DT) to mice. Significant DT-specific mucosal IgA antibody (Ab) and serum IgG, IgA and IgM Ab responses were induced when LPS-depleted mCT E112K or native CT (nCT) was co-administered nasally with DT. The analysis of DT-specific Ab-forming cell (AFC) responses supported the Ab titers and significant numbers of DT-specific IgA AFC were present in the lungs, nasal passages and submandibular glands. Furthermore, DT-specific IgG AFC in cervical lymph nodes (CLN) and the spleen were induced in mice administered with DT nasally with either mCT or nCT. The analysis of antigen-specific T cell responses revealed that increased DT-specific CD4+ T cell proliferative and Th2-type cytokine responses were induced in mice nasally-immunized with DT and the LPS-free form of mCT. The neutralization of diphtheria toxin by Abs showed that DT-specific IgG Ab responses in serum and lung lavages of mice immunized with DT and mCT were protective. Furthermore, it was shown that an IgA-enriched fraction of lung lavages possessed diphtheria toxin-specific neutralizing activity. These results are the first demonstration that nasally co-administered mCT E112K can induce DT-specific protective Ab responses in mucosal compartments (e.g. lung lavages and the lungs).     

Chitosan microparticles for mucosal vaccination against diphtheria: oral and nasal efficacy studies in mice

In this study, the ability of chitosan microparticles to enhance both the systemic and local immune responses against diphtheria toxoid (DT) after oral and nasal administration in mice was investigated.Firstly, DT was associated to chitosan microparticles to determine antigen loading and release. Then DT loaded chitosan microparticles, DT in phosphate buffered saline (PBS) and empty chitosan microparticles (as controls) were fed intragastrically and administered nasally to mice. Mice were also subcutaneously immunised with DT associated with alum. All mice were vaccinated in week 1 and boosted in week 3. Sera were analysed for anti-DT IgG and nasal washings and faeces for anti-DT IgA titres using an enzyme linked immunosorbent assay.Loading capacities of about 25% and loading efficacies of about 100% were obtained after loading the chitosan microparticles with DT. No DT was released at 37 degrees C in PBS. Compared to intragastrical feeding with DT in PBS, a strong enhancement of the systemic and local immune responses against DT were found in mice fed with DT loaded chitosan microparticles. In addition, a dose-dependent immune reaction was observed for mice vaccinated with different doses of DT associated to chitosan microparticles. Significant systemic humoral immune responses were also found after nasal vaccination with DT associated to chitosan microparticles.DT associated to chitosan microparticles results in protective systemic and local immune response against DT after oral vaccination, and in significant enhancement of IgG production after nasal administration. Hence, these in vivo experiments demonstrate that chitosan microparticles are very promising mucosal vaccine delivery systems.     

Intranasal immunization of BALB/c mice with enterotoxigenic Escherichia coli colonization factor CS6 encapsulated in biodegradable poly(DL-lactide-co-glycolide) microspheres

Mice were intranasally administered enterotoxigenic Escherichia coli colonization factor CS6 encapsulated in poly(DL-lactide-co-glycolide) microspheres (CS6-PLG), with immune response measured and compared to that of similarly administered native CS6 and CS6 plus mutant heat-labile enterotoxin mucosal adjuvant (CS6+mLT). Native CS6 and the CS6-PLG microspheres administered intranasally to mice induced serum IgG responses, with the CS6-PLG microspheres inducing a significantly greater (P<0.001) response than native CS6. Following intranasal administration of native CS6, no fecal IgG and IgA responses were measured; however, the CS6-PLG microspheres induced significantly greater (P<0.001) fecal IgG and IgA responses than native CS6. The coadministration of the mLT mucosal adjuvant with CS6 induced significantly greater serum (P<0.001) and fecal (P<0.01) responses than the CS6-PLG microspheres. However, following intranasal administration of the mLT adjuvant, the mice showed definite signs of distress, indicating an adverse reaction to the mLT. Thus, this brings into question the safety of the mLT and its use as an intranasal adjuvant. In contrast, the PLG-microspheres administered intranasally caused no noticeable distress to the mice. The results obtained in this study indicate that the encapsulation of CS6 in PLG-microspheres administered intranasally to mice acted in an adjuvant capacity to enhance the CS6 immune response.     

Protection against influenza virus infection by intranasal administration of C3d-fused hemagglutinin

For the induction of mucosal immune responses by intranasal vaccination, cholera toxin B subunits (CTB) and Escherichia coli heat-labile toxin (LT) are often administered as mucosal adjuvants in order to enhance immune responses to mucosally co-administered bystander antigens. However, these toxin also are the causative agents of diarrhea. There is a demand for the establishment of an effective and safer adjuvant or vaccine that elicits mucosal immunity, but does not require the use of CTB or LT adjuvants. In order to induce protective mucosal immune responses in the nasal area against influenza virus infection, we have examined the recombinant protein composed of the complement component, C3d, which is fused to the secreted form of hemagglutinin (sHA-mC3d3) in the influenza-BALB/c mouse model. The fusion protein sHA-mC3d3, the secretory form of hemagglutinin, and the transmembrane form of HA (tmHA) from the influenza virus were intranasally administered to the mice with or without CTB containing a trace amount of holotoxin (CTB*) as an adjuvant. After intranasal administration of these proteins with CTB*, all mice produced nasal IgA and serum IgG antibodies (Abs) against the viral HA. In addition, viral infection was completely inhibited in these mice. In contrast, in the absence of the adjuvant, only sHA-mC3d3-induced locally secreted IgA and serum IgG Abs and provided complete protection against the influenza virus challenge. Thus, C3d fused to the influenza HA antigen is an effective and safe tool for mucosal vaccination.     

Iscom and iscom-matrix enhance by intranasal route the IgA responses to OVA and rCTB in local and remote mucosal secretions.

Iscoms, with rCTB incorporated via the GM1 receptor, enhanced in mice the mucosal immunogenicity of rCTB as antigen after intranasal (i.n.) administration both by inducing IgA response in the remote intestinal tract mucosa and by a 100-fold increase of the specific IgA locally in the lungs. Iscom-matrix as a separate entity mixed with rCTB enhanced the rCTB-IgA response similarly. While OVA in iscoms induced high mucosal IgA responses, iscom-matrix co-administered with OVA induced low or no mucosal IgA response to OVA. A synergism between iscoms and rCTB could only be seen as an adjuvant targeting effect enhancing the IgA response to OVA in the remote genital tract mucosa. In serum, the immunomodulatory effect of iscoms after i.n. administration was seen as an enhanced serum IgG2a response.     

Intranasal immunization of mice with recombinant Streptococcus gordonii expressing NadA of Neisseria meningitidis induces systemic bactericidal antibodies and local IgA

NadA and NhhA, two surface proteins of serogroup B Neisseria meningitidis identified as candidate vaccine antigens, were expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant strains were used to immunize BALB/c mice by the intranasal route and the local and systemic immune response was assessed. Mice were inoculated with recombinant bacteria administered alone or with LTR72, a partially inactivated mutant of Escherichia coli heat-labile enterotoxin, as a mucosal adjuvant. Intranasal immunization with live bacteria expressing NadA induced a significant serum antibody response, with a prevalence of the IgG2a subclass, bactericidal activity in the sera of 71% of animals, and a NadA-specific IgA response in nasal and bronchoalveolar lavages. A formalin-inactivated recombinant strain of S. gordonii expressing NadA was also administered intranasally, inducing a systemic and mucosal humoral response comparable to that of live bacteria. The administration of recombinant bacteria with the mucosal adjuvant LTR72 stimulated a stronger systemic antibody response, protective in 85% of sera, while did not increase the local IgA response. Recombinant S. gordonii expressing NhhA induced a systemic but not mucosal antibody response. These data support the role of NadA as vaccine candidate against serogroup B meningococci, and the use of S. gordonii as vector for intranasal vaccination.     

Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination

The antibody responses to influenza virus A/PR/8/34 HA vaccine and protection against virus challenge in mice given the vaccine together with the B subunit of cholera toxin (CTB) intranasally were compared with those in mice given the vaccine with CTB perorally, intraperitoneally or subcutaneously. Intranasal vaccination induced remarkably higher levels of antiviral IgA antibodies in both respiratory washings and serum than did other routes of vaccination. The titres of antiviral IgG antibodies in respiratory washings and serum, and haemagglutination-inhibiting (HI) antibodies in serum, were similar after intranasal and parenteral vaccination. Oral vaccination, however, induced low levels of antiviral IgG antibodies but no detectable HI antibodies. Moreover, intranasal immunization elicited significantly higher titres of antiviral IgA antibodies in intestinal secretions in comparison with oral immunization. In contrast, parenteral immunization failed to induce these IgA antibodies. In virus challenge studies, a greater protective effect was seen after intranasal and intraperitoneal vaccination than after other routes of vaccination. These results suggest that intranasal inoculation of combined HA vaccine and CTB is superior to oral or parenteral inoculation in protecting mice. Furthermore, the intestinal antiviral IgA responses suggest that intranasal administration of CTB-combined vaccines could be effective not only against respiratory pathogens but also against enteropathogens.     

Induction of pulmonary mucosal immune responses with a protein vaccine targeted to the DEC-205/CD205 receptor

It is of great interest to develop a pneumonic plague vaccine that would induce combined humoral and cellular immunity in the lung. Here we investigate a novel approach based on targeting of dendritic cells using the DEC-205/CD205 receptor (DEC) via the intranasal route as way to improve mucosal cellular immunity to the vaccine. Intranasal administration of Yersinia pestis LcrV (V) protein fused to anti-DEC antibody together with poly IC as an adjuvant induced high frequencies of IFN-γ secreting CD4⁺ T cells in the airway and lung as well as pulmonary IgG and IgA antibodies. Anti-DEC:LcrV was more efficient to induce IFN-γ/TNF-α/IL-2 secreting polyfunctional CD4⁺ T cells when compared to non-targeted soluble protein vaccine. In addition, the intranasal route of immunization with anti-DEC:LcrV was associated with improved survival upon pulmonary challenge with the virulent CO92 Y. pestis. Taken together, these data indicate that targeting dendritic cells via the mucosal route is a potential new avenue for the development of a mucosal vaccine against pneumonic plague.     

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.     

Intranasal vaccination induced cross-protective secretory IgA antibodies against SARS-CoV-2 variants with reducing the potential risk of lung eosinophilic immunopathology

To control the coronavirus disease 2019 (COVID-19) pandemic, there is a need to develop vaccines to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. One candidate is a nasal vaccine capable of inducing secretory IgA antibodies in the mucosa of the upper respiratory tract, the initial site of infection. However, regarding the development of COVID-19 vaccines, there is concern about the potential risk of inducing lung eosinophilic immunopathology as a vaccine-associated enhanced respiratory disease as a result of the T helper 2 (Th2)-dominant adaptive immune response. In this study, we investigated the protective effect against virus infection induced by intranasal vaccination of recombinant trimeric spike protein derived from SARS-CoV-2 adjuvanted with CpG oligonucleotides, ODN2006, in mouse model. The intranasal vaccine combined with ODN2006 successfully induced not only systemic spike-specific IgG antibodies, but also secretory IgA antibodies in the nasal mucosa. Secretory IgA antibodies showed high protective ability against SARS-CoV-2 variants (Alpha, Beta and Gamma variants) compared to IgG antibodies in the serum. The nasal vaccine of this formulation induced a high number of IFN-γ-secreting cells in the draining cervical lymph nodes and a lower spike-specific IgG1/IgG2a ratio compared to that of subcutaneous vaccination with alum as a typical Th2 adjuvant. These features are consistent with the induction of the Th1 adaptive immune response. In addition, mice intranasally vaccinated with ODN2006 showed less lung eosinophilic immunopathology after viral challenge than mice subcutaneously vaccinated with alum adjuvant. Our findings indicate that intranasal vaccine adjuvanted with ODN2006 could be a candidate that can prevent the infection of antigenically different variant viruses, reducing the risk of vaccine-associated enhanced respiratory disease.     

Human antibody response to fragments A and B of diphtheria toxin and a synthetic peptide of amino acid residues 141-157 of fragment A

Examination of a selection of serum samples from adults from two regions of England showed that 50% of men in the 16-24 years and over 55 years age groups had high titres of antibody to diphtheria toxin (DT). In contrast, only 11% of women aged 16 to over 55 years had high titres of antibody to DT. All human antisera with high anti-DT titres reacted with a synthetic peptide (SP) corresponding to the amino acids 141-157 of DT fragment A, with sera from men aged 35 to over 55 years showing the highest titres. High antibody titres to fragment A paralleled those to SP in both sexes. Titres of antibody to DT fragment B were highest in individuals with high titres to DT. In sera from both sexes immunoglobulin G1 was the predominant subclass reactive with all three antigens. However, both IgG1 and IgG4 and to a lesser extent IgG2 and IgG3 were present in immunoglobulin concentrates.     

Humoral immune responses by prime-boost heterologous route immunizations with CTB-MPR(649-684), a mucosal subunit HIV/AIDS vaccine candidate

CTB-MPR(649-684) is a translational fusion protein consisting of the cholera toxin B subunit and a 36-residue peptide, MPR(649-684), corresponding to the conserved membrane proximal ectodomain of gp41. CTB-MPR(649-684) was previously shown to induce HIV-1 transcytosis-blocking antibodies in mice. In this report, we describe an effective immunization regimen for this novel anti HIV-1 vaccine-candidate. Bacterially-produced CTB-MPR(649-684) was intranasally and/or intraperitoneally administered to investigate several prime-boost heterologous route immunization regimens. Mucosal priming with the adjuvant cholera toxin elicited significant levels of vaginal IgA and serum IgG specific to MPR(649-684). Systemic boosting after mucosal priming enhanced the levels of serum and mucosal antibodies. Systemic priming induced a strong serum anti-MPR(649-684) IgG response, which was efficiently recalled and augmented by either systemic or mucosal boosting. However, this regimen was less effective in inducing secretory anti-MPR(649-684) IgA. The serum anti-MPR(649-684) IgG subtype profile revealed that both IgG1 and IgG2a were induced in all the immunization regimens, and that mucosal co-administration of cholera toxin shifted the bias to the latter subtype. We concluded that, of the various immunization regimens examined here, mucosal priming with adjuvant followed by systemic boosting exhibited the best response in respect to either systemic or mucosal anti-MPR(649-684) antibodies. Most importantly, mucosal antibodies elicited by this regimen significantly inhibited HIV-1 transcytosis in a human tight epithelium model.