Pseudomonas aeruginosa PAO1 outer membrane vesicles-diphtheria toxoid conjugate as a vaccine candidate in a murine burn model.

Pseudomonas aeruginosa is an opportunistic pathogen considered a common cause of nosocomial infection with high morbidity and mortality in burn patients. Immunoprophylaxis techniques may lower the mortality rate of patients with burn wounds infected by P. aeruginosa; consequently, this may be an efficient strategy to manage infections caused by this bacterium. Several pathogenic Gram-negative bacteria like P. aeruginosa release outer membrane vesicles (OMVs), and structurally OMV consists of several antigenic components capable of generating a wide range of immune responses. Here, we evaluated the immunogenicity and efficacy of P. aeruginosa PA-OMVs (PA-OMVs) conjugated with the diphtheria toxoid (DT) formulated with alum adjuvant (PA-OMVs-DT + adj) in a mice model of burn wound infection. ELISA results showed that in the group of mice immunized with PA-OMVs-DT + adj conjugated, there was a significant increase in specific antibodies titer compared to non-conjugated PA-OMVs or control groups. In addition, the vaccination of mice with PA-OMVs-DT + adj conjugated generated greater protective effectiveness, as seen by lower bacterial loads, and eightfold decreased inflammatory cell infiltration with less tissue damage in the mice burn model compared to the control group. The opsonophagocytic killing results confirmed that humoral immune response might be critical for PA-OMVs mediated protection. These findings suggest that PA-OMV-DT conjugated might be used as a new vaccine against P. aeruginosa in burn wound infection.

Subcutaneous immunoglobulin replacement for treatment of humoral immune dysfunction in patients with chronic lymphocytic leukemia.

BACKGROUND: Patients with chronic lymphocytic leukemia (CLL) experience hypogammaglobinemia and non-neutropenic infections. In this exploratory proof of concept study, our objective was to determine the prevalence of humoral immunodeficiency in patients with CLL and serum IgG ≥ 400 mg/dL, and to evaluate the efficacy of subcutaneous immunoglobulin (SCIG) in this population. PATIENTS AND METHODS: Patients with CLL with serum IgG ≥ 400 mg/dL were evaluated for serum IgG, IgM, IgA, along with pre/post vaccine IgG titers to diphtheria, tetanus, and Streptococcus pneumoniae. Patients with evidence of humoral dysfunction were treated with SCIG with Hizentra every 7±2 days for 24 weeks. RESULTS: Fifteen patients enrolled with median IgG = 782 mg/dL [IQR: 570 to 827], and 6/15 (40%) responded to vaccination with Td, while 5/15 (33%) responded to vaccination with PPV23. 14/15 (93.3%) demonstrated humoral immunodeficiency as evidenced by suboptimal vaccine responses, and were treated with SCIG. In patients treated with SCIG, serum IgG increased from 670 mg/dL [IQR: 565 to 819] to 1054 mg/dL [IQR: 1040 to 1166] after 24 weeks (95% CI: 271-540). For streptococcus pneumoniae, the median protective serotypes at baseline was 8 [IQR: 4 to 9] and increased to 17 [IQR: 17 to 19] after 24 weeks (95% CI: 6.93-13.72). Non-neutropenic infections (NNI) decreased from 14 to 5 during treatment with SCIG. CONCLUSIONS: Patients with CLL demonstrate humoral immunodeficiency despite IgG > 400 mg/dL. For these patients, SCIG is well tolerated and efficacious in improving serum IgG, specific IgG to streptococcus pneumoniae, and may decrease reliance on antibiotics for the treatment of NNIs. CLINICAL TRIALS REGISTRATION: NCT03730129.

Seroprevalence of diphtheria toxoid IgG antibodies in the Malaysian population.

BACKGROUND: Despite high childhood immunization coverage, sporadic cases of diphtheria have been reported in Malaysia in recent years. This study aims to evaluate the seroprevalence of diphtheria among the Malaysian population. METHODS: A total of 3317 respondents age 2 years old to 60 years old were recruited in this study from August to November 2017. Enzyme-linked immunosorbent assay (ELISA) was used to measure the level of IgG antibody against the toxoid of C. diphtheriae in the blood samples of respondents. We classified respondent antibody levels based on WHO definition, as protective (≥0.1 IU/mL) and susceptible (< 0.1 IU/mL) to C. diphtheriae infection. RESULTS: Among the 3317 respondents, 57% were susceptible (38.1% of children and 65.4% of adults) and 43% (61.9% of children and 34.6% of adults) had protective antibody levels against diphtheria. The mean antibody level peaked among individuals aged 1-2 years old (0.59 IU/mL) and 6-7 years old (0.64 IU/mL) but generally decreased with age, falling below 0.1 IU/mL at around 4-6 years old and after age 20 years old. There was a significant association between age [Children: χ2 = 43.22(df = 2),p < 0.001)], gender [Adults: χ2 = 5.58(df = 1),p = 0.018] and ethnicity [Adults: χ2 = 21.49(df = 5),p = 0.001] with diphtheria toxoid IgG antibody level. CONCLUSIONS: About 57% of the Malaysian population have inadequate immunity against diphtheria infection. This is apparently due to waning immunity following childhood vaccination without repeated booster vaccination in adults. Children at age 5-6 years old are particularly vulnerable to diphtheria infection. The booster vaccination dose normally given at 7 years should be given earlier, and an additional booster dose is recommended for high-risk adults.

Characterisation of diphtheria monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Immunoassays are used for routine potency assessment of several vaccines, in some cases having been specifically developed as alternatives to in vivo potency tests. These methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against diphtheria with a view to select antibodies that can be used for development of an in vitro potency immunoassay for diphtheria vaccines. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined by a cell-based toxin neutralisation test and diphtheria toxin-domain recognition was determined by Western blotting. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Replacing the in vivo toxin challenge test with an in vitro assay for assessment of potency for diphtheria toxoid containing vaccines.

Replacement of the potency tests for diphtheria vaccines is a high priority for the international initiative to reduce, refine, and replace animal use in vaccine testing. Diphtheria toxoid containing vaccine products marketed in the US currently require potency testing by the United States Public Health Service (USPHS) test, which includes an in vivo passive protection test with a diphtheria toxin challenge. Here we describe an in vitro Diphtheria Vero Cell (DVC) assay which combines the immunization approach from the USPHS test and the use of a cell based neutralization assay for serological testing of vaccine potency. The DVC assay reduces the overall number of animals used compared to other serological potency tests and eliminates the in vivo toxin challenge used in the US test. The DVC assay can be used to test vaccine products with a low or high diphtheria toxoid dose. It has been optimized and validated for use in a quality control testing environment. Results demonstrate similar sera antibody unitage as well as agreement between the serum neutralization values determined using the USPHS test and the DVC assay and thus support the use of the DVC assay for routine and stability testing for diphtheria toxoid containing vaccine products.

Induction of antibody responses in mice immunized intranasally with Type I interferon as adjuvant and synergistic effect of chitosan.

Type I IFNs are a range of host-derived molecules with adjuvant potential; they have been used for many years in the treatment of cancer and viral hepatitis. Therefore, the safety of IFNs for human use has been established. In this study, we evaluated the mucosal adjuvanticity of IFN-ß administered intranasally to mice with diphtheria toxoid, and suggested a method to improve its adjuvanticity. When IFN-ß alone was used as a mucosal adjuvant, no clear results were obtained. However, simultaneous administration of IFN-ß and chitosan resulted in an enhancement of the specific serum immunoglobulin G (IgG) and IgA antibody responses, the mucosal IgA antibody response, and antitoxin titers. Furthermore, the intranasal administration of IFN-α alone resulted in a greater increase in antibody titer than IFN-ß, and a synergistic effect with chitosan was also observed. These findings suggest that intranasal administration of chitosan and Type I IFNs may display an effective synergistic mucosal adjuvant activity.

Identification of Formaldehyde-Induced Modifications in Diphtheria Toxin.

Diphtheria toxoid is produced by detoxification of diphtheria toxin with formaldehyde. This study was performed to elucidate the chemical nature and location of formaldehyde-induced modifications in diphtheria toxoid. Diphtheria toxin was chemically modified using 4 different reactions with the following reagents: (1) formaldehyde and NaCNBH3, (2) formaldehyde, (3) formaldehyde and NaCNBH3 followed by formaldehyde and glycine, and (4) formaldehyde and glycine. The modifications were studied by SDS-PAGE, primary amino group determination, and liquid chromatography-electrospray mass spectrometry of chymotryptic digests. Reaction 1 resulted in quantitative dimethylation of all lysine residues. Reaction 2 caused intramolecular cross-links, including the NAD+-binding cavity and the receptor-binding site. Moreover, A fragments and B fragments were cross-linked by formaldehyde on part of the diphtheria toxoid molecules. Reaction 3 resulted in formaldehyde-glycine attachments, including in shielded areas of the protein. The detoxification reaction typically used for vaccine preparation (reaction 4) resulted in a combination of intramolecular cross-links and formaldehyde-glycine attachments. Both the NAD+-binding cavity and the receptor-binding site of diphtheria toxin were chemically modified. Although CD4+ T-cell epitopes were affected to some extent, one universal CD4+ T-cell epitope remained almost completely unaltered by the treatment with formaldehyde and glycine.

[Diphtheria: a 'forgotten' disease?] / Difterie: een 'vergeten' ziekte?

Respiratory diphtheria is an acute respiratory tract infection. The high mortality rates (5-10%) are related to airway obstruction and local and systemic effects of the diphtheria toxin. Vaccination against diphtheria has been available through the Dutch national vaccination programme since the 1950s. The disease has now become rare as a result of herd immunity by these vaccinations. As a consequence, the disease has largely been forgotten, which can result in it not being recognised and treated in time. In addition, diphtheria antitoxin is not always available. In this article, we are drawing attention to the need for immunisation. We also look back at how diphtheria prevention started in the Netherlands.

Further investigations of the IgE response to tetanus and diphtheria following covaccination with acellular rather than cellular Bordetella pertussis.

BACKGROUND: It has previously been shown in an uncontrolled study that the IgE response to vaccine antigens is downregulated by co-vaccination with cellular Bordetella pertussis vaccine. METHODS: In the present study, we compared in a controlled trial the humoral immune response to diphtheria toxoid (D) and tetanus toxoid (T) in relation to co-vaccinated cellular or acellular B pertussis vaccine. IgE, IgG4, and IgG to D and T were analyzed at 2, 7, and 12 months of age in sera of children vaccinated with D and T (DT, N = 68), cellular (DTPw, N = 68), 2- or 5-component acellular B pertussis vaccine (DTPa2, N = 64; DTPa5, N = 65). RESULTS: One month after vaccination, D-IgE was detected in 10% sera of DTPw-vaccinated children, whereas vaccination in the absence of whole-cell pertussis resulted in 50%-60% IgE positivity. Six months after vaccination, the IgE antibody levels were found to be more persistent than the IgG antibodies. These diphtheria findings were mirrored by those for tetanus. Only minor differences between vaccine groups were found with regard to D-IgG and T-IgG. No immediate-type allergic reactions were observed. CONCLUSION: Cellular (but not acellular) B pertussis vaccine downregulates IgE to co-vaccinated antigens in infants. We assume that the absence of immediate-type allergic reactions is due to the high levels of IgG antibodies competing with IgE antibodies.

Analysis of the Amino Acid Sequence Variation of the 67-72p Protein and the Structural Pili Proteins of Corynebacterium diphtheriae for their Suitability as Potential Vaccine Antigens.

The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 67-72p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 67-72p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 67-72p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells.The aim of this study was to identify the potential vaccine antigens in Corynebacterium diphtheriae strains by in silico analysis of the amino acid variation in the 67­72p surface protein that is involved in the colonization and induction of epithelial cell apoptosis in the early stages of infection. The analysis of pili structural proteins involved in bacterial adherence to host cells and related to various types of infections was also performed. A polymerase chain reaction (PCR) was carried out to amplify the genes encoding the 67­72p protein and three pili structural proteins (SpaC, SpaI, SapD) and the products obtained were sequenced. The nucleotide sequences of the particular genes were translated into amino acid sequences, which were then matched among all the tested strains using bioinformatics tools. In the last step, the affinity of the tested proteins to major histocompatibility complex (MHC) classes I and II, and linear B-cell epitopes was analyzed. The variations in the nucleotide sequence of the 67­72p protein and pili structural proteins among C. diphtheriae strains isolated from various infections were noted. A transposition of the insertion sequence within the gene encoding the SpaC pili structural proteins was also detected. In addition, the bioinformatics analyses enabled the identification of epitopes for B-cells and T-cells in the conserved regions of the proteins, thus, demonstrating that these proteins could be used as antigens in the potential vaccine development. The results identified the most conserved regions in all tested proteins that are exposed on the surface of C. diphtheriae cells.

Stabilised aluminium phosphate nanoparticles used as vaccine adjuvant.

Aluminium phosphate is a commonly used adjuvant consisting of heterogeneously sized aggregates up to several micrometers. However, aluminium phosphate nanoparticles may exhibit an improved adjuvant effect. In this study, nanoparticles were made by sonication of commercially available aluminium phosphate adjuvant, resulting in particles with a size (Z-average diameter) between 200-300 nm and a point of zero charge of 4.5. To prevent reaggregation, which occurred within 14 days, a screening of excipients was performed to identify stabilisers effective under physiological conditions (pH 7.4, 290 mOsm). The amino acids threonine, asparagine, and L-alanyl-L-1-aminoethylphosphonic acid (LAPA) stabilised sonicated aluminium phosphate. Particle sizes remained stable between 400-600 nm at 37 °C during 106 days. Contrarily, arginine induced strong reaggregation to a particle size larger than 1000 nm. The stability of aluminium phosphate nanoparticles was strongly affected by the pH. Aggregation mainly occurred below pH 7. The adsorption capacity, a potentially relevant parameter for adjuvants, was slightly reduced in the presence of asparagine, when using a model antigen (lysozyme). LAPA, arginine, threonine and aspartic acid reduced protein adsorption significantly. The adjuvant effect of aluminium phosphate nanoparticles was studied by immunisation of mice with diphtheria toxoid adjuvanted with the aluminium phosphate nanoparticles. The presence of LAPA, threonine, aspartic acid or asparagine did not alter diphtheria toxoid-specific antibody or toxin-neutralising antibody titres. Arginine increased diphtheria toxoid-specific antibody titres but not toxin-neutralising antibody titres. In conclusion, aluminium phosphate nanoparticles were stabilised by particular amino acids and induced an adjuvant effect comparable to that of aluminium phosphate microparticles.

Risk of Measles and Diphtheria Introduction and Transmission on Bonaire, Caribbean Netherlands, 2018.

Endemic transmission of measles has been reestablished in Venezuela, and outbreaks of diphtheria remain ongoing across Latin America (LA). Hence, a large cross-sectional population-based serosurveillance study was conducted on Bonaire, one of the Dutch Leeward Antilles, to assess specific age and population groups at risk. Participants (aged 0-90 years) donated a blood sample and completed a questionnaire (n = 1,129). Antibodies against measles and diphtheria were tested using bead-based multiplex immunoassays. Our data revealed that immunity against measles is suboptimal, especially for those aged less than 5 years from Suriname, Aruba, and former Dutch Antilles (SADA), and adolescents from LA; and against diphtheria for persons aged more than 30 years, particularly among females and residents from SADA and LA. As refugees arrive persistently, health authorities on the Dutch Leeward Antilles should be on alert to detect early cases and prevent subsequent transmission. Ultimately, there is an urgent need for serosurveillance studies in the Caribbean region.

Proteomics of diphtheria toxoid vaccines reveals multiple proteins that are immunogenic and may contribute to protection of humans against Corynebacterium diphtheriae.

Introduced for mass immunization in the 1920s, vaccines against diphtheria are among the oldest and safest vaccines known. The basic principle of their production is the inactivation of purified diphtheria toxin by formaldehyde cross-linking, which converts the potentially fatal toxin in a completely harmless protein aggregate, which is still immunogenic. Since in addition to diphtheria toxin also other proteins may be secreted by Corynebacterium diphtheriae during cultivation, we assumed that diphtheria toxoid might not be the only component present in the vaccine. To address this question, we established a protocol to reverse formaldehyde cross-linking and carried out mass spectrometric analyses. Different secreted, membrane-associated and cytoplasmic proteins of C. diphtheriae were detected in several vaccine preparations from across the world. Based on these results, bioinformatics and Western blot analyses were applied to characterize if these proteins are immunogenic and may therefore support protection against C. diphtheriae. In frame of this study, we could show that the C. diphtheriae toxoid vaccines induce antibodies against different C. diphtheriae proteins and against diphtheria toxin secreted by Corynebacterium ulcerans, an emerging pathogen which is outnumbering C. diphtheriae as cause of diphtheria-like illness in Western Europe.

Persistence of bactericidal antibodies 4 years after a booster dose of quadrivalent meningococcal diphtheria toxoid conjugate vaccine (MenACWY-D).

One dose of quadrivalent meningococcal conjugate vaccine (MenACWY) was first recommended for US adolescents (ages 11-12 years) in 2005 to protect against invasive meningococcal disease (IMD). In 2010, after evidence emerged about waning protection within 5 years after MenACWY vaccination, the US Advisory Committee on Immunization Practices (ACIP) recommended a MenACWY booster at age 16 years. We used a serum bactericidal assay with human complement (hSBA) to evaluate antibody persistence after a MenACWY-D booster in a sample of 110 participants who received the booster 4 years earlier in a phase 2 study. High proportions (89.9-98.2%) of participants maintained hSBA titers (≥1:4) associated with protection against IMD; a majority (81.7-97.2%) also had hSBA titers ≥1:8, a more conservative threshold. These findings support ACIP recommendations regarding MenACWY booster vaccination, which are aimed at protecting adolescents and young adults throughout the period in which they are at increased risk of IMD.

Adult Diphtheria Vaccination.

Low adult vaccination coverage in Indonesia may contribute to a recent outbreak of diphtheria in Indonesia. Although well known as a pediatric vaccine, diphtheria vaccination should be administered as booster to adolescence and adults for longer prevention. Adult vaccine differs from pediatric vaccine but have similar protection. Additionally, there is special recommendation to vaccinate pregnant women and elderly people aged 65 years or more.

Reactogenicity and immunogenicity of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (Tdap) in pregnant and nonpregnant women.

OBJECTIVE: Tetanus toxoid, reduced diphtheria toxoid, and acellular pertusiss (Tdap) vaccine is recommended during each pregnancy, regardless of prior receipt. Data on reactogenicity and immunogenicity, particularly after repeated Tdap, are limited. We compared local injection-site and systemic reactions and serologic response following Tdap in (1) pregnant and nonpregnant women and (2) pregnant women by self-reported prior Tdap receipt. STUDY DESIGN: Pregnant women (gestational age 20-34 weeks) and nonpregnant women receiving Tdap were enrolled in this observational study. Injection-site and systemic reactions were assessed for one week post-vaccination. Pertussis toxin, filamentous hemagglutinin, pertactin, fimbriae, tetanus and diphtheria specific IgG antibody titers were determined by standardized enzyme-linked immunosorbent assay at baseline and 28 days post-vaccination. Reactogenicity and serologic responses were compared by pregnancy status, and within pregnant women by self-reported prior Tdap receipt. RESULTS: 374 pregnant and 225 nonpregnant women were vaccinated. Severe local or systemic reactions or "any" fever were uncommon (≤3% for both groups). Moderate/severe injection-site pain was significantly higher in pregnant (17.9%) versus nonpregnant (11.1%) women, but did not prompt a healthcare visit. Proportions of other moderate/severe or any severe reactions were not significantly higher in pregnant compared to nonpregnant women. Moderate/severe (including pain) and severe reactions were not significantly higher in pregnant women receiving repeat versus first-time Tdap. Antibody titers increased from baseline to post-vaccination for all vaccine antigens in pregnant and nonpregnant women; post-vaccination titers against pertussis toxin and filamentous hemagglutinin were significantly higher in nonpregnant versus pregnant women (p < 0.01). CONCLUSION: Tdap was well-tolerated in pregnant and nonpregnant women. Pregnant women were more likely to report moderate/severe pain at the Tdap injection-site compared with nonpregnant women, but did not necessitate medical visits. Prior Tdap receipt did not increase occurrence of moderate/severe local or systemic reactions in pregnant women. Serologic responses to all vaccine antigens were robust. Clinical Trial Registration@ClinicalTrials.gov. NCT02209623. https://clinicaltrials.gov/ct2/show/NCT02209623.

Coated and Hollow Microneedle-Mediated Intradermal Immunization in Mice with Diphtheria Toxoid Loaded Mesoporous Silica Nanoparticles.

PURPOSE: To examine the immunogenicity of diphtheria toxoid (DT) loaded mesoporous silica nanoparticles (MSNs) after coated and hollow microneedle-mediated intradermal immunization in mice. METHODS: DT was loaded into MSNs and the nanoparticle surface was coated with a lipid bilayer (LB-MSN-DT). To prepare coated microneedles, alternating layers of negatively charged LB-MSN-DT and positively charged N-trimethyl chitosan (TMC) were coated onto pH-sensitive microneedle arrays via a layer-by-layer approach. Microneedle arrays coated with 5 or 3 layers of LB-MSN-DT were used to immunize mice and the elicited antibody responses were compared with those induced by hollow microneedle-injected liquid formulation of LB-MSN-DT. Liquid DT formulation with and without TMC (DT/TMC) injected by a hollow microneedle were used as controls. RESULTS: LB-MSN-DT had an average size of about 670 nm and a zeta potential of -35 mV. The encapsulation efficiency of DT in the nanoparticles was 77%. The amount of nano-encapsulated DT coated onto the microneedle array increased linearly with increasing number of the coating layers. Nano-encapsulated DT induced stronger immune responses than DT solution when delivered intradermally via hollow microneedles, but not when delivered via coated microneedles. CONCLUSION: Both the nano-encapsulation of DT and the type of microneedles affect the immunogenicity of the antigen.

GM-CSF improves the immune response to the diphtheria-component in a multivalent vaccine.

Multivalent tetanus and diphtheria toxoid containing vaccines belong to the most frequently applied vaccines. However, there is an imbalance in the degree of protection against the two antigens with insufficient long-term protection against diphtheria, particularly in the elderly population. We have previously reported a positive correlation between granulocyte macrophage-colony stimulating factor (GM-CSF) and the production of diphtheria-specific antibodies. Therefore, in the present study we analyzed the effects of in vivo applied recombinant GM-CSF on immunization with multivalent tetanus/diphtheria vaccine in mice of different age. In vivo application of GM-CSF lead to enhanced production of diphtheria-specific antibodies as well as more diphtheria-specific CD4+ T cells following vaccination with multivalent tetanus/diphtheria vaccine. In contrast, the humoral and cellular immune response to the tetanus component was unaltered. Furthermore, application of GM-CSF resulted in more splenic CD11b+ dendritic cells (DCs) with a higher MHC-II expression. GM-CSF also induced a stronger recruitment of CD11b+ DCs to the injected muscle. Most remarkably, GM-CSF was able to boost the diphtheria-specific immune response to the multivalent vaccine in aged mice. This study demonstrates that local administration of GM-CSF is able to improve immune responsiveness to the diphtheria component of multivalent tetanus/diphtheria vaccine in young and old mice. This information could be useful for the future design of vaccines for the elderly.

Immunogenicity of diphtheria toxoid and poly(I:C) loaded cationic liposomes after hollow microneedle-mediated intradermal injection in mice.

In this study, we aimed to investigate the immunogenicity of cationic liposomes loaded with diphtheria toxoid (DT) and poly(I:C) after hollow microneedle-mediated intradermal vaccination in mice. The following liposomal formulations were studied: DT loaded liposomes, a mixture of free DT and poly(I:C)-loaded liposomes, a mixture of DT-loaded liposomes and free poly(I:C), and liposomal formulations with DT and poly(I:C) either individually or co-encapsulated in the liposomes. Reference groups were DT solution adjuvanted with or without poly(I:C) (DT/poly(I:C)). The liposomal formulations were characterized in terms of particle size, zeta potential, loading and release of DT and poly(I:C). After intradermal injection of BALB/c mice with the formulations through a hollow microneedle, the immunogenicity was assessed by DT-specific ELISAs. All formulations induced similar total IgG and IgG1 titers. However, all the liposomal groups containing both DT and poly(I:C) showed enhanced IgG2a titers compared to DT/poly(I:C) solution, indicating that the immune response was skewed towards a Th1 direction. This enhancement was similar for all liposomal groups that contain both DT and poly(I:C) in the formulations. Our results reveal that a mixture of DT encapsulated liposomes and poly(I:C) encapsulated liposomes have a similar effect on the antibody responses as DT and poly(I:C) co-encapsulated liposomes. These findings may have implications for future design of liposomal vaccine delivery systems.