Cross-reactivity and avidity of antibody responses induced in humans by the oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX

We investigated whether the oral inactivated, multivalent enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of four E. coli strains over-expressing the colonisation factors (CFs) CFA/I, CS3, CS5 and CS6, combined with the toxoid LCTBA, could induce cross-reactive antibodies to CFs related to the CFA/I and CS5 families. We also evaluated the avidity of vaccine induced antibodies against the toxoid and CFs.Cross-reactivity was analysed in mucosal (faecal and antibodies in lymphocyte supernatants, ALS) samples, and antibody avidity in serum and ALS samples, from two phase I trials: a primary vaccination study, where two oral doses of ETVAX were given ± the double mutant heat labile toxin (dmLT) adjuvant at a 2-week interval, and a booster vaccination study, where a single booster dose of ETVAX was given 13–23 months after primary vaccinations.We found that 65–90% of subjects who had responded to CFA/I in ALS or faecal specimens also developed cross-reactive antibodies to the related CFs tested, i.e. CS1, CS14 and CS17, and that approximately 80% of those responding to CS5 also responded to the closely related CS7. For subjects who had developed cross-reactive antibodies, the magnitudes of responses against vaccine CFs and related non-vaccine CFs were comparable. Using both a simple method of antibody avidity determination based on limiting antigen dilution, as well as a chaotropic ELISA method, we found that the avidity of serum and ALS antibodies to key vaccine antigens increased after a late booster dose compared to after primary vaccination.Our results suggest that the cross-reactive antibody responses against multiple CFs may result in expanded ETEC strain coverage of ETVAX and that repeated vaccinations induce vaccine-specific antibodies with increased binding capacity.     

Safety and immunogenicity of an improved oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine administered alone and together with dmLT adjuvant in a double-blind,randomized, placebo-controlled Phase I study

BackgroundWe have developed a new oral vaccine against enterotoxigenic Escherichia coli (ETEC), which is the most common cause of bacterial diarrhea in children in developing countries and in travelers.MethodsThe vaccine was tested for safety and immunogenicity alone and together with double-mutant heat-labile toxin (dmLT) adjuvant in a double-blind, placebo-controlled Phase I study in 129 Swedish adults. The vaccine consists of four inactivated recombinant E. coli strains overexpressing the major ETEC colonization factors (CFs) CFA/I, CS3, CS5, and CS6 mixed with an LT B-subunit related toxoid, LCTBA. Volunteers received two oral doses of vaccine alone, vaccine plus 10 μg or 25 μg dmLT or placebo. Secretory IgA antibody responses in fecal samples and IgA responses in secretions from circulating intestine-derived antibody secreting cells were assessed as primary measures of vaccine immunogenicity.ResultsThe vaccine was safe and well tolerated; adverse events were few and generally mild with no significant differences between subjects receiving placebo or vaccine with or without adjuvant. As many as 74% of subjects receiving vaccine alone and 83% receiving vaccine plus 10 μg dmLT showed significant mucosal IgA responses to all five primary vaccine antigens and about 90% of all vaccinees responded to at least four of the antigens. Subjects receiving vaccine plus 10 μg dmLT responded with significantly increased intestine-derived anti-CS6 responses compared to subjects receiving vaccine alone.ConclusionsThe vaccine was safe and broadly immunogenic. dmLT further enhanced mucosal immune responses to CF antigens present in low amounts in the vaccine. Based on these encouraging results, the vaccine will be tested for safety and immunogenicity in different age groups including infants in Bangladesh and for protective efficacy in travelers.     

Kinetics of antibody-secreting cell and fecal IgA responses after oral cholera vaccination in different age groups in a cholera endemic country

Immune responses to oral enteric vaccines in children and infants may be influenced by factors such as age, previous priming with related microorganisms and breast feeding. In this study, we aimed to determine optimal time points to assess immune responses to oral enteric vaccines in different clinical specimens. This was done by investigating antibody secreting cell (ASC) and fecal antibody responses on different days after vaccination using the licensed oral cholera vaccine Dukoral, containing cholera toxin B-subunit (rCTB) and inactivated Vibrio cholerae bacteria, as a model vaccine.Two vaccine doses were given 2 weeks apart to infants (6–11 months), young children (12–18 months), toddlers (19 months–5 years) and adults in a cholera endemic country (Bangladesh). IgA ASC responses, as determined by the antibodies in lymphocyte supernatant (ALS) assay, plasma IgA and IgG responses and secretory IgA (SIgA) responses in extracts of fecal samples were evaluated 4/5 and 7 days after each vaccination.After the first vaccine dose, anti-CTB ALS IgA responses in adults and toddlers were high and comparable on day 5 and 7, while responses were low and infrequent in young children. After the second dose, highest ALS responses were detected on day 5 among the time points studied in all age groups and the responses declined until day 7. In contrast, plasma IgA and IgG anti-CTB responses were high both on day 5 and 7 after the second dose. Fecal SIgA responses in young children and infants were highest on day 7 after the second dose.Our results suggest that ASC/ALS responses to two doses of the oral cholera vaccine Dukoral and related oral vaccines should be analyzed earlier than previously recommended (day 7) at all ages. Fecal antibody responses should preferably be analyzed later than ASC/ALS responses to detect the highest antibody responses.     

Evaluation of the safety and immunogenicity of the oral inactivated multivalent enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi adults in a double-blind,randomized, placebo-controlled Phase I trial using electrochemiluminescence and ELISA assays for immunogenicity analyses

The safety and immunogenicity of the second generation oral enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, consisting of inactivated recombinant E. coli strains over-expressing the colonization factors (CFs) CFA/I, CS3, CS5 and CS6 and the heat labile toxoid LCTBA, were evaluated in Bangladeshi volunteers. To enable analysis of antibody responses against multiple vaccine antigens for subsequent use in small sample volumes from children, a sensitive electrochemiluminescence (ECL) assay for analysis of intestine-derived antibody-secreting cell responses using the antibodies in lymphocyte secretions (ALS) assay was established using Meso Scale Discovery technology.Three groups of Bangladeshi adults (n = 15 per group) received two oral doses of ETVAX with or without double mutant LT (dmLT) adjuvant or placebo in the initial part of a randomized, double-blind, placebo-controlled, age-descending, dose-escalation trial. CF- and LTB-specific ALS and plasma IgA responses were analyzed by ECL and/or ELISA.ETVAX was safe and well tolerated in the adults. Magnitudes of IgA ALS responses determined by ECL and ELISA correlated well (r = 0.85 to 0.98 for the five primary antigens, P < 0.001) and ECL was selected as the ALS readout method. ALS IgA responses against each of the primary antigens were detected in 87–100% of vaccinees after the first and in 100% after the second vaccine dose. Plasma IgA responses against different CFs and LTB were observed in 62–93% and 100% of vaccinees, respectively. No statistically significant adjuvant effect of dmLT on antibody responses to any antigen was detected, but the overall antigenic breadth of the plasma IgA response tended to favor the adjuvanted vaccine when responses to 4 or more or 5 vaccine antigens were considered. Responses in placebo recipients were infrequent and mainly detected against single antigens.The promising results in adults supported testing ETVAX in descending age groups of children.ClinicalTrials.gov Identifier: NCT02531802.     

On birth single dose live attenuated OPV and BCG vaccination induces gut cathelicidin LL37 responses at 6 week of age: A natural experiment

In a cross sectional study, we show that infants who received single dose of live attenuated OPV and BCG vaccines within 48 h of birth, have higher excretion of human cathelicidin LL37 (p < 0.05) in stool at 6 wk of age. This response remained unchanged in multivariate analysis after adjusting for sex, mode of delivery, infant age, mother age birth weight and breast milk feeding pattern. This analysis also reveals that irrespective of vaccination, girl infants have higher human-beta-defencin2 (HBD2) and exclusively breastfed infants have higher total and anti-polio specific IgA to all three subtypes in stool (p < 0.05). However, vaccination induces anti-polio IgA responses only to infants who are exclusively breastfed. Thus on-birth live attenuated vaccination may provide non-specific beneficial effect against infections while exclusive breastfeeding enhance protection by boosting vaccine induced IgA. The result also suggests that in polio endemic area, exclusive breastfeeding may be sufficient for mucosal anti-polio responses during early infancy.     

Safety and immunogenicity of a parenterally administered rotavirus VP8 subunit vaccine in healthy adults

BackgroundThe P2-VP8 subunit vaccine for the prevention of rotavirus gastroenteritis is comprised of a truncated VP8 subunit protein from the rotavirus Wa strain (G1[P8]) fused to the tetanus toxin P2 epitope, and adsorbed on aluminum hydroxide for intramuscular administration.MethodsThree groups of 16 adults were randomized to receive three injections of P2-VP8 (12) or placebo (4) at doses of 10, 30 or 60 μg of vaccine. IgG and IgA antibodies to P2-VP8 were assessed by ELISA in serum and lymphocyte supernatant (ALS). Serum samples were tested for neutralizing antibodies to homologous and heterologous strains of rotavirus.ResultsThe vaccine was well-tolerated. All vaccine recipients demonstrated significant IgA responses and all but one demonstrated IgG responses; in the 60 μg cohort, geometric mean titers (GMTs) rose 70- and 80-fold for IgA and IgG, respectively. Homologous neutralizing antibody responses were observed in about half of participants in all three dose cohorts; in the 60 μg cohort, GMTs against Wa rose from 128 to 992. Neutralizing antibody responses were robust to P[8] strains, moderate to P[4] strains and negligible to P[6] strains. ALS IgA responses were dose dependent.ConclusionsThe P2-VP8 subunit vaccine was well tolerated and evoked promising immune responses.Clinical trials registrationNCT01764256     

Effect of infectious bursal disease (IBD) vaccine on Salmonella Enteritidis infected chickens

BackgroundChickens infected with both infectious bursal disease virus (IBDV) and Salmonella had higher mortality. In this work, we investigated the effect of IBDV vaccine (modified live-virus bursal disease vaccine, Nobilis strain 228E®) on experimentally infected chickens with Salmonella Enteritidis (SE).MethodsFour experimental groups were included in this study, negative control group, 228E®group, 228E® + SE infected group, and SE infected group. Chickens were ocularly administrated 228E® at 12 days of age and orally infected with S. Enteritidis at 13 days of age. Sera, intestinal fluid, blood, cloacal swabs and tissue samples were collected at 1, 2 and 3 weeks post vaccination (PV).ResultsThe recorded mortalities were higher in the 228E® + SE infected group, compared to the SE infected group. The anti-S. Enteritidis serum antibody titer and the intestinal mucosal IgA level were higher in the SE infected group at 2 and 3 weeks PV, compared to 228E® + SE infected group. S. Enteritidis fecal shedding and organ colonization were significantly higher in the 228E® + SE infected group than the SE infected group at 2 and 3 weeks PV. The 228E® + SE group had significantly lower bursa to body weight ratios at 2 and 3 weeks PV, as well as had higher bursal lesion scores than the SE infected group. IBDV vaccine depressed the specific-SE systemic and mucosal antibody responses, but did not affect the specific-SE cellular immune responses.ConclusionChickens administrated IBDV vaccine, followed by S. Enteritidis infection, could cause a significant effect on the bursa of Fabricius, resulting in failure of systemic and mucosal antibody responses to the S. Enteritidis and reduce the elimination and the clearance of S. Enteritidis.     

Exonic single nucleotide polymorphisms within TLR3 associated with infant responses to serogroup C meningococcal conjugate vaccine

The introduction of the serogroup C meningococcal (MenC) conjugate vaccination has successfully controlled the burden of disease associated with this serogroup in many countries. However, considerable inter-individual variation is observed in immune responses to MenC vaccine, and little is understood of the determinants of this variability. Previously, we reported an association between single nucleotide polymorphisms (SNPs) in TLR3 and CD44 and the persistence of MenC vaccine immunity. Here we further examine polymorphisms within these two candidate genes and immune responses to MenC vaccine. MenC-specific IgG concentrations and serum bactericidal assay (SBA) titres were measured one month after a primary course of MenC vaccination in 318 human infants. Tagging SNPs (TagSNPs) within TLR3 and CD44 were genotyped and regional imputations carried out to screen these genes for variations associated with immunological responses to MenC vaccine. This study reports an association between an exonic variant (rs3775290, P = 0.025) in TLR3 and MenC IgG concentrations, as well as an association between three SNPs in CD44 (rs3794109, P = 0.021; rs3794110, P = 0.022; rs112762, P = 0.049) and MenC SBA titres. These data support our previous findings of an association between SNPs in TLR3 and CD44, and present novel findings implicating exonic variants in these genes with MenC vaccine responses.     

Immune responses following meningococcal serogroups A,C, Y and W polysaccharide vaccination in C2-deficient persons: Evidence for increased levels of serum bactericidal antibodies

Complement C2 deficiency (C2D) is associated with immunological diseases and increased susceptibility to invasive infections caused by encapsulated bacteria such as Neisseria menigitidis. In this study we evaluate the immunogenicity of vaccination against N. menigitidis in C2D.C2D patients (n = 22) and controls (n = 52) were given a tetravalent meningococcal polysaccharide vaccine. Serum bactericidal antibody (SBA) titres (serogroups A, C, Y and W) were analysed using a rabbit complement source. Levels of IgG, IgM, and IgA, factor B, and factor H, polymorphisms of MBL and Fc-gamma receptors were determined.The C2D patients responded with an increased SBA titre to all four serogroups (p < 0.001). The response rates define as SBA titres ≥8 were found to be between 85.7% and 92.5%. The post-vaccination titres for serogroups C, Y and W were equal to healthy controls. C2D patients with a history of invasive infection had a lower post-vaccination SBA titres both compared to healthy C2D persons (p = 0.03) and compared to controls (p < 0.0001). We found that the G2M*n/G2M*n genotype were associated with a higher SBA titres after immunization (p = 0.03). None of the other investigated immunological factors appear to be important in influencing the vaccine responses. Autoimmune diseases in C2D did not affect the vaccine response.In general, vaccination against meningococci gave rise to antibody responses in the C2D patients that equal healthy controls. The response rate was lower to serogroup A and among C2D patients with history of invasive infections. The presence of G2M*n/G2M*n genotype was associated with higher SBA titres after immunization.     

BAFF,stimulatory DNA and IL-15 stimulates IgA memory B cells and provides a novel approach for analysis of memory responses to mucosal vaccines

Assessment of immune responses induced by mucosal vaccines is to a large extent based on measurement of IgA levels in mucosal secretions and detection of short-lived effector IgA-secreting cells circulating in peripheral blood. Since these immunological parameters poorly reflect long-term IgA-mediated responses, we sought to investigate novel approaches that would enable detection of vaccine specific IgA memory B cells. We demonstrate that stimulation of human peripheral blood mononuclear cells in vitro with immunostimulatory DNA in combination with B cell-activating factor (BAFF) and IL-15 promotes differentiation of IgA memory B cells to IgA-secreting cells. By using the inactivated oral cholera vaccine Dukoral^® we demonstrate that vaccine specific IgA memory B cells are induced by oral immunization and are circulating for at least 9 months after vaccination. We also show that stimulated IgA memory B cells do not secrete IgA unless they reencounter the specific antigen.     

First-in-human trial of the post-exposure tuberculosis vaccine H56:IC31 in Mycobacterium tuberculosis infected and non-infected healthy adults

BackgroundH56:IC31 is a candidate tuberculosis vaccine comprising a fusion protein of Ag85B, ESAT-6 and Rv2660c, formulated in IC31 adjuvant. This first-in-human, open label phase I trial assessed the safety and immunogenicity of H56:IC31 in healthy adults without or with Mycobacterium tuberculosis (M.tb) infection.MethodsLow dose (15 μg H56 protein in 500 nmol IC31) or high dose (50 μg H56, 500 nmol IC31) vaccine was administered intramuscularly thrice, at 56-day intervals. Antigen-specific T cell responses were measured by intracellular cytokine staining and antibody responses by ELISA.ResultsOne hundred and twenty-six subjects were screened and 25 enrolled and vaccinated. No serious adverse events were reported. Nine subjects (36%) presented with transient cardiovascular adverse events. The H56:IC31 vaccine induced antigen-specific IgG responses and Th1 cytokine-expressing CD4⁺ T cells. M.tb-infected vaccinees had higher frequencies of H56-induced CD4⁺ T cells than uninfected vaccinees. Low dose vaccination induced more polyfunctional (IFN-γ⁺TNF-α⁺IL-2⁺) and higher frequencies of H56-specific CD4⁺ T cells compared with high dose vaccination. A striking increase in IFN-γ-only-expressing CD4⁺ T cells, displaying a CD45RA⁻CCR7⁻ effector memory phenotype, emerged after the second high-dose vaccination in M.tb-infected vaccinees. TNF-α⁺IL-2⁺ H56-specific memory CD4⁺ T cells were detected mostly after low-dose H56 vaccination in M.tb-infected vaccinees, and predominantly expressed a CD45RA⁻CCR7⁺ central memory phenotype. Our results support further clinical testing of H56:IC31.     

Robust mucosal-homing antibody-secreting B cell responses induced by intramuscular administration of adjuvanted bivalent human norovirus-like particle vaccine

BackgroundTwo major antigenically heterogenous norovirus genogroups (GI and GII) commonly infect humans and are the leading cause of foodborne, viral gastrointestinal infections in adults.MethodsWe assessed B cell responses in participants in a double-blind, placebo-controlled, dose-escalation phase 1 study of the safety and immunogenicity of an intramuscular bivalent norovirus virus-like particle (VLP) vaccine. The vaccine contained a GI.1 VLP (Norwalk) and a consensus GII.4 VLP, representing the two major genotypes that cause human disease, and was administered on days 0 and 28 to healthy adults aged 18–49 years. Four separate cohorts received increasing doses of 5 μg, 15 μg, 50 μg, and 150 μg of each VLP adjuvanted in monophosphoryl lipid A and alum. PBMCs were analyzed for B cell activation and mucosal homing markers (flow cytometry) and VLP-specific and total IgG and IgA Ab-secreting cells (ASCs); and serum titers of VLP-specific IgG, IgA, and Pan-Ig were determined.ResultsThe vaccine elicited CD27+ CD38+ plasmablasts and high frequencies of ASCs specific for both VLP antigens in the peripheral blood at 7 days after the first dose. The plasmablasts exhibited a mucosal-homing phenotype and included a high proportion of IgA ASCs. Serum antibodies increased as early as 7 days after the first immunization.ConclusionsThe data suggest that a single dose of the IM bivalent norovirus vaccine is effective in activating pre-existing B cell memory. The rapid B cell response and the mucosal homing phenotype of induced ASCs are consistent with anamnestic responses in subjects primed by prior oral norovirus infection.This study is registered at ClinicalTrials.gov Identifier NCT01609257.     

Induction of mucosal and systemic immune responses against the common O78 antigen of an oral inactivated ETEC vaccine in Bangladeshi children and infants

We tested an oral enterotoxigenic Escherichia coli (ETEC) vaccine, ETVAX, consisting of inactivated E. coli overexpressing the most prevalent ETEC colonization factors (CFs) and a toxoid (LCTBA), in Bangladeshi children for capacity to induce mucosal and plasma immune responses against O78 lipopolysaccharide (LPS) expressed on the vaccine strains. The vaccine was given ± double-mutant heat-labile toxin (dmLT) adjuvant. We evaluated the impact of dmLT on anti-O78 LPS immune responses and whether such responses can predict responses against the CFs as a marker for vaccine “take”. Two fractionated doses of ETVAX ± different amounts of dmLT were administered biweekly to groups of children 24–59 (n = 125), 12–23 (n = 97) and 6–11 (n = 158) months of age. Immune responses were evaluated in antibody in lymphocyte supernatants (ALS), fecal extracts and plasma. ALS IgA responses against O78 LPS were induced in 44–49% of the children aged 12–59 months. The magnitudes of the ALS responses were significantly higher in children receiving a half-dose (5 × 10¹⁰ bacteria) of ETVAX ± dmLT than in placebo recipients. <10% of the vaccinees aged 6–11 months mounted ALS responses against O78 LPS. However, 49% of the infants developed fecal secretory IgA responses which were significantly more frequent in those receiving a quarter-dose (2.5 × 10¹⁰ bacteria) of vaccine + dmLT (62%) compared to a quarter-dose alone (36%). Plasma IgA antibody responses were induced in 80% of older children and 36% of infants. The frequencies of O78 LPS responses in plasma and feces were comparable or higher than against the vaccine CFs in infants. Our findings show that ETVAX induced mucosal and systemic immune responses against O78 LPS in all age groups and that dmLT improved intestinal immune responses among infants. These observations may have implications for more successful use of other oral vaccines based on O antigens in children.     

Salivary antibody levels in adolescents in response to a meningococcal serogroup C conjugate booster vaccination nine years after priming: systemically induced local immunity and saliva as potential surveillance tool

BackgroundIn several countries large-scale immunization of children and young adults with Meningococcal serogroup C (MenC) conjugate vaccines has induced long-standing herd protection. Salivary antibodies may play an important role in mucosal protection against meningococcal acquisition and carriage.AimTo investigate antibody levels in (pre)adolescents primed 9 years earlier with a single dose of MenC-polysaccharide tetanus toxoid conjugated (MenC-TT) vaccine and the response to a booster vaccination, with special focus on age-related differences and the relation between salivary and serum antibody levels.MethodsNine years after priming, healthy 10- (n = 91), 12- (n = 91) and 15-year-olds (n = 86) received a MenC-TT booster vaccination. Saliva and serum samples were collected prior to and 1 month and 1 year after vaccination. MenC-polysaccharide(MenC-PS)-specific antibody levels were measured using a fluorescent-bead-based multiplex immunoassay.ResultsBefore the booster, MenC-PS-specific IgG and IgA levels in saliva and serum were low and correlated with age at priming. The booster induced a marked increase in salivary MenC-PS-specific IgG (>200-fold), but also in IgA (∼10-fold). One year after the booster, salivary IgG and IgA had remained above pre-booster levels in all age groups (∼20-fold and ∼3-fold, respectively), with persistence of highest levels in the 15-year-olds. MenC-PS-specific IgG and IgA levels in saliva strongly correlated with the levels in serum.ConclusionParenteral MenC-TT booster vaccination induces a clear increase in salivary MenC-PS-specific IgG and IgA levels and persistence of highest levels correlates with age. The strong correlation between serum and salivary antibody levels indicate that saliva may offer an easy and reliable tool for future antibody surveillance.     

Humoral,T-cell and B-cell immune responses to seasonal influenza vaccine in solid organ transplant recipients receiving anti-T cell therapies

BackgroundWe analyzed the impact of the anti-T-cell agents basiliximab and antithymocyte globulins (ATG) on antibody and cell-mediated immune responses after influenza vaccination in solid-organ transplant recipients.Methods71 kidney and heart transplant recipients (basiliximab [n = 43] and ATG [n = 28]) received the trivalent influenza vaccine. Antibody responses were measured at baseline and 6 weeks post-vaccination by hemagglutination inhibition assay; T-cell responses were measured by IFN-γ ELISpot assays and intracellular cytokine staining (ICS); and influenza-specific memory B-cell (MBC) responses were evaluated using ELISpot.ResultsMedian time of vaccination from transplantation was 29 months (IQR 8–73). Post-vaccination seroconversion rates were 26.8% for H1N1, 34.1% for H3N2 and 4.9% for influenza B in the basiliximab group and 35.7% for H1N1, 42.9% for H3N2 and 14.3% for influenza B in the ATG group (p = 0.44, p = 0.61, and p = 0.21, respectively). The number of influenza-specific IFN-γ-producing cells increased significantly after vaccination (from 35 to 67.5 SFC/10⁶ PBMC, p = 0.0007), but no differences between treatment groups were observed (p = 0.88). Median number of IgG-MBC did not increase after vaccination (H1N1, p = 0.94; H3N2 p = 0.34; B, p = 0.79), irrespective of the type of anti-T-cell therapy.ConclusionsAfter influenza vaccination, a significant increase in antibody and T-cell immune responses but not in MBC responses was observed in transplant recipients. Immune responses were not significantly different between groups that received basiliximab or ATG.     

Preparation and evaluation of antigen/N-trimethylaminoethylmethacrylate chitosan conjugates for nasal immunization

The frequent outbreak of respiratory infectious diseases such as influenza and pulmonary tuberculosis calls for new immunization strategies with high effectiveness. Nasal immunization is one of the most potential methods to prevent the diseases infected through the respiratory tract. In this study, we designed a water-soluble system based on antigen/N-trimethylaminoethylmethacrylate chitosan conjugates for nasal immunization. N-trimethylaminoethylmethacrylate chitosan (TMC) was synthesized by free radical polymerization of chitosan and N-trimethylaminoethylmethacrylate chloride and identified by ¹H NMR and FT-IR. Thiolated ovalbumin (OVA) was covalently conjugated to maleimide modified TMC with high conjugation efficiency. OVA conjugated TMC (OVA–TMC) significantly increased uptake of OVA by Raw 264.7 cells, which was 2.38 times higher than that of OVA/TMC physical mixture (OVA + TMC) at 4 h. After nasal administration, OVA–TMC showed higher transport efficiency to superficial and deep cervical lymph nodes than OVA + TMC or OVA alone. Balb/C mice were intranasally given with OVA–TMC three times at 2-week internals to evaluate the immunological effect. The serum IgG, IgG1 and IgG2a levels of the OVA–TMC group were 17.9–87.9 times higher than that of the OVA + TMC group and comparable to that of the intramuscular group. The secretory IgA levels in nasal wash and saliva of the OVA–TMC group were 5.2–7.1 times higher than that of the OVA + TMC group while the secretory IgA levels of the intramuscular alum-precipitated OVA group were not increased. After immunofluorescence staining of nasal cavity, IgA antibody secreting cells were mainly observed in the lamina propria regions and glands of nasal mucosa. OVA–TMC showed little toxicity to the nasal epithelia or cilia of rats after nasal administration for three consecutive days. These results demonstrated that antigen conjugated TMC can induce both systemic and mucosal immune responses after nasal administration and may serve as a convenient, safe and effective vaccine for preventing respiratory infectious diseases.     

Methotrexate reduces vaccine-specific immunoglobulin levels but not numbers of circulating antibody-producing B cells in rheumatoid arthritis after vaccination with a conjugate pneumococcal vaccine

BackgroundTreatment with methotrexate (MTX) in patients with rheumatoid arthritis (RA) leads to decreased total immunoglobulin (Ig) levels and impairs vaccine-specific IgG antibody levels following pneumococcal vaccination. The mechanisms by which MTX exerts these effects in RA are unknown. We aimed to evaluate whether MTX reduces vaccine-specific serum Ig levels and their functionality in RA patients following vaccination with pneumococcal conjugate vaccine, and if numbers of antigen-specific circulating plasmablasts are affected.MethodsTen patients with RA on MTX and 10 RA patients without disease modifying anti-rheumatic drug (DMARD) were immunized with a dose 13-valent pneumococcal conjugate vaccine (Prevenar13). Circulating plasmablasts producing total IgG and IgA as well as specific IgG and IgA against two pneumococcal capsular serotypes (6B and 23F) were enumerated using ELISPOT 6 days after vaccination. IgG levels against both these serotypes were determined with ELISA before and 4–6 weeks after vaccination. Positive antibody response was defined as ⩾2-fold increase of pre-vaccination antibody levels. The functionality of vaccine specific antibodies to serotype 23F was evaluated by measuring their ability to opsonize bacteria using opsonophagocytic assay (OPA) in 4 randomly chosen RA patients on MTX and 4 RA patients without DMARD.ResultsAfter vaccination, RA patients on MTX showed significant increase in pre- to postvaccination antibody levels for 6B (p < 0.05), while patients without DMARD had significant increases for both 6B and 23F (p < 0.05 and p < 0.01, respectively). Only 10% of RA on MTX and 40% of RA patients without DMARD showed positive post-vaccination antibody responses for both serotypes. Increased opsonizing ability after vaccination was detected in 1 of 4 RA patients on MTX and 3 of 4 patients on RA without DMARD. However, numbers of circulating total and vaccine-specific IgG- or IgA-producing plasmablasts did not differ between RA patients with or without MTX.ConclusionsMTX treatment in RA leads to reduced vaccine-specific antibody responses and their functionality compared to untreated RA following pneumococcal vaccination using polysaccharide-protein conjugate vaccine. However, since there was no reduction in numbers of circulating total or vaccine-specific antibody-producing plasmablasts after vaccination this effect is probably not due to reduced activation of B cells in lymphoid tissue.Clinical trial registration: NCT02240888.     

Lasting immune memory against hepatitis B following challenge 10–11 years after primary vaccination with either three doses of hexavalent DTPa-HBV-IPV/Hib or monovalent hepatitis B vaccine at 3, 5 and 11–12 months of age

BackgroundThe combined hexavalent diphtheria–tetanus–pertussis–hepatitis B-inactivated poliomyelitis – Haemophilus influenzae type b conjugate vaccine (Infanrix hexa™; DTPa-HBV-IPV/Hib: GlaxoSmithKline Vaccines) induces robust responses to the HBV component when administered at 3, 5 and 11–12 months of age. We assessed long term HBV antibody persistence 10–11 years after primary vaccination in infancy.MethodsAntibody persistence and immune memory were assessed post-primary vaccination at 3, 5, 11–12 months with DTPa-HBV-IPV/Hib, or monovalent HBV vaccine (Engerix™ B, GlaxoSmithKline Vaccines) co-administered with DTPa-IPV/Hib (Infanrix™-IPV/Hib, GlaxoSmithKline Vaccines) in 185 children aged 11–12 years. Blood samples were collected before and 1 month after a challenge dose of Engerix™ B (10 μg dose).Results10–11 years after primary vaccination the percentage of subjects with persisting anti-HBs antibody concentrations ≥10 mIU/ml was 48.4% in the DTPa-HBV-IPV/Hib group and 58.4% in the DTPa-IPV/Hib + HBV group. After the HBV challenge dose, the percentage with anti-HBs ≥100 mIU/ml increased from 14.7% to 93.6% in the DTPa-HBV-IPV/Hib group and 19.1% to 94.4% in the DTPa-IPV/Hib + HBV group. Anti-HBs GMCs increased by at least 187-fold in each group. An anamnestic response (≥4-fold increase in initially seropositive or anti-HBs concentration ≥10 mIU/ml in initially seronegative subjects) was observed in 96.8% and 96.6% of subjects in the DTPa-HBV-IPV/Hib and DTPa-IPV/Hib + HBV groups, respectively. No serious adverse events occurred that were considered related to challenge vaccination.ConclusionAdministration of HBV as part of a combination vaccine or as a monovalent vaccine induced long lasting immune memory against HBV in children primed at 3, 5 and 11 months of age. Antibody persistence and immune memory were similar, suggesting that protection afforded by DTPa-HBV-IPV/Hib and monovalent HBV vaccines, is likely to be of similar duration. The administration of HBV challenge dose 10–11 years after the 3, 5, 11–12 months primary schedule induced strong anamnestic responses and was well tolerated.This study is registered at www.clinicaltrials.gov NCT01138098.     

Consecutive inoculations of influenza virus vaccine and poly(I:C) protects mice against homologous and heterologous virus challenge

Mucosal immunity induced through natural infection by influenza virus has potent cross-protective activity, compared to subcutaneous vaccination-induced systemic immunity. Compared to natural infection with influenza virus, however, a single intranasal vaccination with an inactivated influenza virus vaccine and poly(I:C) is not sufficient to induce primary immune response in naïve animals. The reasons for this moderate effect are not fully understood. Here, we demonstrated that intranasal vaccination with formalin-inactivated influenza virus vaccine and poly(I:C) for five consecutive days elicits high levels of virus-specific nasal IgA and serum IgG responses, while vaccination without poly(I:C) induced little response. Mice immunized with influenza virus vaccine and poly(I:C) for five consecutive days sustained high levels of virus-specific IgA in nasal wash and IgG in serum until at least 6 months after vaccination. Furthermore, intranasal vaccination with influenza virus vaccine and poly(I:C) protected mice against homologous and heterologous influenza virus challenge. These results suggest that consecutive inoculations of influenza virus vaccine and poly(I:C) is an alternative method to induce primary immune responses in naïve subjects.     

Tetanus Toxoid carrier protein induced T-helper cell responses upon vaccination of middle-aged adults

IntroductionVaccines frequently induce suboptimal immune responses in the elderly, due to immunological ageing. Timely vaccination may be a strategy to overcome this problem, which classifies middle-aged adults as an interesting target group for future vaccine interventions. However, the immunological fitness of the middle-aged population is ill-defined. It is currently unknown whether effective T-cell help towards B-cells is initiated by conjugate-carrier vaccines at middle-age.AimWe characterized systemic Tetanus Toxoid (TT) specific T-helper cell responses in the circulation of middle-aged adults (50–65 years of age, n = 31) having received the MenACWY-TT vaccination.MethodsBlood samples were taken pre- as well as 7 days, 28 days, and 1 year post-vaccination. TT-specific T-cell responses were determined by IFNγ Elispot and by the secretion of IFNγ, IL13, IL10, IL17, and IL21 in cell culture supernatants. Circulating CD4+CXCR5+ICOS+IL21+ cells were analyzed by flow cytometry, and meningococcal and TT-specific IgG responses by bead-based immunoassays. The correlation between the T-cell help and humoral responses was evaluated.ResultsVaccination with a TT-carrier protein induced a mixed TT-specific Th1 (IFNγ), Th2 (IL13, IL10), and Th17 (IL17) response in most participants. Additionally, circulating CD4+CXCR5+ICOS+IL21+ cells were significantly increased 7 days post-vaccination. Pre-vaccination TT-specific cytokine production and post-vaccination Th2 responses correlated positively with the increase of CD4+CXCR5+ICOS+IL21+ cells. No correlation between T-cell help and antibody responses was found.ConclusionThe characteristics of the T-cell response upon a TT-carrier vaccination suggests effective T-cell help towards B-cells in response to meningococcal polysaccharides, although the absence of a correlation with the antibody responses warrants further clarification. However, the robust T-helper cell response in middle-aged adults, decades after previous TT vaccinations, strengthens the classification of this age group for future vaccine interventions in the context of population ageing.