Specific memory B cell response and participation of CD4+ central and effector memory T cells in mice immunized with liposome encapsulated recombinant NE protein based Hepatitis E vaccine candidate

BackgroundLiposome encapsulated neutralizing epitope protein of Hepatitis E virus (HEV), rNEp, our Hepatitis E vaccine candidate, was shown to be immunogenic and safe in pregnant and non-pregnant mice and yielded sterilizing immunity in rhesus monkeys.MethodsThe current study in Balb/c mice assessed the levels and persistence of anti-HEV IgG antibodies by ELISA, frequencies of B, memory B, T and memory T cells by flow cytometry and HEV-specific IgG secreting memory B cells by ELISPOT till 420 days post immunization (PI) with 5 μg rNEp encapsulated in liposome based adjuvant (2 doses, 4 weeks apart). Mice immunized with a lower dose (1 μg) were assessed only for anamnestic response post booster dose.ResultsVaccine candidate immunized mice (5 μg dose) elicited strong anti-HEV IgG response that was estimated to persist for lifetime. At day 120 PI, frequency of memory B cells was higher in immunized mice than those receiving adjuvant alone. Anti-HEV IgG titers were lower in mice immunized with 1 μg dose. A booster dose yielded a heightened antibody response in mice with both high (>800 GMT, 5 μg) and low (⩽100 GMT, 1 μg) anti-HEV IgG titers. At day 6th post booster dose, HEV-specific antibody secreting plasma cells (ASCs) were detected in 100% and 50% of mice with high and low anti-HEV IgG titers, respectively, whereas the frequencies of CD4⁺ central and effector memory T cells were high in mice with high anti-HEV IgG titers only.ConclusionsTaken together, the vaccine candidate effectively generates persistent and anamnestic antibody response, elicits participation of CD4⁺ memory T cells and triggers memory B cells to differentiate into ASCs upon boosting. This approach of assessing the immunogenicity of vaccine candidate could be useful to explore the longevity of HEV-specific memory response in future HEV vaccine trials in human.     

Pertussis vaccination during pregnancy in Belgium: Follow-up of infants until 1 month after the fourth infant pertussis vaccination at 15 months of age

Vaccination of pregnant women with a pertussis containing vaccine is a recommended strategy in some industrialized countries, to protect young infants from severe disease. One of the effects of the presence of high titers of passively acquired maternal antibodies in young infants is blunting of immune responses to infant vaccination. We present infant immune responses to a fourth pertussis containing vaccine dose at 15 months of age, as a follow-up of previously presented data.In a prospective cohort study, women were either vaccinated with an acellular pertussis vaccine (Boostrix^®) during pregnancy (vaccine group) or received no vaccine (control group).All infants were vaccinated with Infanrix Hexa^® according to the standard Belgian vaccination schedule (8/12/16 weeks, 15 months). We report results from blood samples collected before and 1 month after the fourth vaccine dose. Immunoglobulin G (IgG) antibodies against pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (Prn), tetanus toxoid (TT) and diphtheria toxoid (DT) were measured using commercially available ELISA tests. Antibody levels were expressed in International Units per milliliter.Demographic characteristics were similar in the vaccine and control group. Before the fourth vaccine dose, significantly lower antibody titers were measured in the vaccine group compared to the control group for anti-Prn IgG (p = 0.003) and anti-DT IgG (p = 0.023), with a steep decay of antibody titers since post-primary vaccination. One month after the fourth dose, antibody titers were only significantly lower in the vaccine group for anti-PT IgG (p = 0.006). For all antigens, there was a rise in antibody titer after the fourth vaccine dose.The present results indicate still a minor blunting effect 1 month after a fourth vaccine dose for anti-PT antibodies. However, a good humoral immune response on all measured antigens was elicited in both groups of children. The clinical significance of such blunting effect is yet unknown.Clinicaltrials.gov identifier: NCT01698346.     

Breastfeeding linked to the reduction of both rotavirus shedding and IgA levels after Rotarix® immunization in Mexican infants

We examined potential risk factors on vaccine virus shedding and antibody seroresponse to human rotavirus vaccine (Rotarix) in Mexican infants. Two doses of Rotarix were administered to infants during the first two visits for their routine childhood immunization (∼8 and 15 weeks of age) in Mexico City. Infant’s characteristics and socioeconomic indicators were obtained, including history of long-term feeding practices (exclusively/predominantly breastfed and exclusively/predominantly non-breastfed). Two serum specimens were collected, one during the second rotavirus vaccine visit and one 7 weeks later. Stool specimens were collected between days 4–7 after each of the two rotavirus vaccine doses. Rotavirus IgA and IgG titers in serum were determined by enzyme immunoassays (EIA) and rotavirus shedding in stool was assessed by EIA and confirmed by RT-PCR.The overall rotavirus IgA geometric mean titers (GMT) increased significantly post dose 2 from post dose 1 [176 (95%CI: 113–273) to 335 (238–471); p = 0.020). Infants who were exclusively/predominantly breastfed were less likely to shed vaccine virus in stool than those who were formula-fed (22% vs. 43%, p = 0.016). Infants who were breastfed had lower rotavirus IgA titers than those who were formula-fed after dose 1 [GMT: 145 (84–250) vs. 267 (126–566) p = 0.188] and dose 2 [236 (147–378) vs.578 (367–910), p = 0.007]. Infants who shed vaccine virus post dose 1 had significantly higher serum IgA GMT than those who did not shed [425 (188–965) vs. 150 (84–266), p = 0.038]. Breastfeeding was linked with the reduction of both stool vaccine shedding, and IgA seroresponse. The reduced rotavirus replication in the gut and shedding after dose 1 may explain in part the lower IgA response in serum.     

Parental perceptions of childhood seasonal influenza vaccination in Singapore: A cross-sectional survey

PurposeSeasonal influenza vaccination is recommended in children aged 6–59 months, but little is known about child vaccination coverage and determinants in Asian settings. We report the results of a survey of knowledge, attitudes, practices, and determinants of child influenza vaccination in Singapore.MethodsIn December 2015-March 2016, we conducted a survey of 332 parents of children aged 6 months to 5 years attending pre-schools. We assessed child influenza vaccine coverage and parental knowledge, attitudes, and practices of child influenza vaccination. We used multivariable regression and structural equation models to identify factors associated with child influenza vaccination.ResultsKnowledge about influenza, perceived benefit of vaccination, and willingness to vaccinate were high. However, only 32% of children had ever received influenza vaccine, and only 15% in the past year. Factors independently associated with child influenza vaccination included: being recommended influenza vaccine by a child’s doctor (prevalence ratio (PR) = 2.47, 95% CI: 1.75–3.48); receiving influenza vaccine information from a private general practitioner (PR = 1.47, 95% CI: 1.05–2.04); regularly receiving pre-travel influenza vaccine (PR = 1.64, 95% CI: 1.19–2.25); higher willingness to vaccinate (PR = 1.58, 95% CI:1.24–2.04 per unit increase in willingness score); and feeling well-informed about influenza vaccine (PR = 1.44, 95% CI: 1.04–1.99). Parents who obtained influenza vaccine information from television were less likely to have vaccinated their child (PR = 0.44, 95% CI: 0.23–0.85). Path analysis indicated that being recommended vaccination by a child's doctor increased willingness to vaccinate and self-efficacy (feeling well-informed about influenza vaccine). Median willingness-to-pay for a dose of influenza vaccine was SGD30 (interquartile range: SGD20-SGD50), and was higher in parents of vaccinated compared with unvaccinated children (SGD45 vs SGD30, p = 0.0012).ConclusionKnowledge and willingness to vaccinate was high in this parent population, but influenza vaccine uptake in children was low. Encouraging medical professionals to recommend vaccination of eligible children is key to improving uptake.     

Comparative analysis of influenza A(H3N2) virus hemagglutinin specific IgG subclass and IgA responses in children and adults after influenza vaccination

Two different influenza vaccines are generally used in many countries; trivalent live attenuated influenza vaccine (LAIV3) and trivalent inactivated influenza vaccine (IIV3). Studies comparing the antibody response to IIV3 and LAIV3 commonly investigate the seroprotective response by hemagglutination-inhibition (HI) assay. However, there is limited data regarding comparative analysis of IgG subclass and IgA responses induced by LAIV3 and IIV3.Fifteen children <5 years received 2 doses of LAIV3 while 14 children aged 10–17 years received one dose. In addition, 15 adults were vaccinated with either intranasal LAIV3 or intramuscular IIV3. We analyzed the H3N2 humoral responses by HI assay and the hemagglutinin (HA) specific IgG1, IgG2, IgG3, IgG4 and IgA1 responses by ELISA. Furthermore, we investigated the avidity of induced IgG antibodies.Pre-existing seroprotective HI antibodies were present in adults (73%) previously vaccinated with IIV3. Vaccination resulted in a significant increase in HI titers in all groups, except LAIV3 vaccinated adults. Furthermore, a negative correlation between age and HI titers in LAIV3 vaccinated subjects was observed post-vaccination. LAIV3 in children and IIV3 in adults induced HA-specific IgG1, low IgG3 but no IgG2 or IgG4. Moreover, significant IgA1 responses were only induced in children. Interestingly, IIV3 and LAIV3 induced IgG antibodies with comparable and significantly augmented avidity post-vaccination in children and adults.Our results suggest that age and/or exposure history play a significant role in determining the antibody response.Clinical trial registry: ClinicalTrials.gov NCT01003288 and NCT01866540     

Immunogenicity of pulsatile-release PLGA microspheres for single-injection vaccination

The World Health Organization's Expanded Programme on Immunization has led to a dramatic rise in worldwide vaccination rates over the past 40 years, yet 19.4 million infants remain underimmunized each year. Many of these infants have received at least one vaccine dose but may remain unprotected because they did not receive subsequent booster doses due to logistical challenges. This study aimed to develop injectable controlled release microparticles with kinetics that mimic common vaccine dosing regimens consisting of large antigen doses administered periodically over the course of months in order to eliminate the need for boosters. Sixteen poly(lactic-co-glycolic acid) (PLGA) microsphere formulations containing bovine serum albumin (BSA) as a model vaccine antigen were screened in vitro to determine their respective release kinetics. Three formulations that exhibited desirable pulsatile release profiles were then selected for studying immunogenicity in mice. Two low-dose microsphere formulations induced peak anti-BSA IgG antibody titers of 13.9 ± 1.3 and 13.7 ± 2.2 log₂ compared to 15.5 ± 1.5 log₂ for a series of three bolus injections delivered at 0, 4, and 8 weeks with an equivalent cumulative dose. Similarly, high-dose formulations induced peak antibody titers that were 16.1 ± 2.1 log₂ compared to 17.7 ± 2.2 log₂ for controls. All three microparticle formulations studied in vivo induced peak antibody titers that were statistically similar to bolus controls. These results suggest that pulsatile antigen release from polymeric microparticles is a promising approach for single-injection vaccination, which could potentially reduce the logistical burden associated with immunization in the developing world.     

Multiple efficacy studies of an adenovirus-vectored foot-and-mouth disease virus serotype A24 subunit vaccine in cattle using homologous challenge

The safety and efficacy of an experimental, replication-deficient, human adenovirus-vectored foot-and-mouth disease virus (FMDV) serotype A24 Cruzeiro capsid-based subunit vaccine (AdtA24) was examined in eight independent cattle studies. AdtA24 non-adjuvanted vaccine was administered intramuscularly to a total of 150 steers in doses ranging from approximately 1.0 × 10⁸ to 2.1 × 10¹¹ particle units per animal. No detectable local or systemic reactions were observed after vaccination. At 7 days post-vaccination (dpv), vaccinated and control animals were challenged with FMDV serotype A24 Cruzeiro via the intradermal lingual route. Vaccine efficacy was measured by FMDV A24 serum neutralizing titers and by protection from clinical disease and viremia after challenge. The results of eight studies demonstrated a strong correlation between AdtA24 vaccine dose and protection from clinical disease (R² = 0.97) and viremia (R² = 0.98). There was also a strong correlation between FMDV A24 neutralization titers on day of challenge and protection from clinical disease (R² = 0.99). Vaccination with AdtA24 enabled differentiation of infected from vaccinated animals (DIVA) as demonstrated by the absence of antibodies to the FMDV nonstructural proteins in vaccinates prior to challenge. Lack of AdtA24 vaccine shedding after vaccination was indicated by the absence of neutralizing antibody titers to both the adenovector and FMDV A24 Cruzeiro in control animals after co-mingling with vaccinated cattle for three to four weeks. In summary, a non-adjuvanted AdtA24 experimental vaccine was shown to be safe, immunogenic, consistently protected cattle at 7 dpv against direct, homologous FMDV challenge, and enabled differentiation of infected from vaccinated cattle prior to challenge.     

Safety and immunogenicity of heterologous prime-boost immunization with viral-vectored malaria vaccines adjuvanted with Matrix-M™

The use of viral vectors in heterologous prime-boost regimens to induce potent T cell responses in addition to humoral immunity is a promising vaccination strategy in the fight against malaria. We conducted an open-label, first-in-human, controlled Phase I study evaluating the safety and immunogenicity of Matrix-M adjuvanted vaccination with a chimpanzee adenovirus serotype 63 (ChAd63) prime followed by a modified vaccinia Ankara (MVA) boost eight weeks later, both encoding the malaria ME-TRAP antigenic sequence (a multiple epitope string fused to thrombospondin-related adhesion protein). Twenty-two healthy adults were vaccinated intramuscularly with either ChAd63-MVA ME-TRAP alone (n = 6) or adjuvanted with 25 μg (n = 8) or 50 μg (n = 8) Matrix-M. Vaccinations appeared to be safe and generally well tolerated, with the majority of local and systemic adverse events being mild in nature. The addition of Matrix-M to the vaccine did not increase local reactogenicity; however, systemic adverse events were reported more frequently by volunteers who received adjuvanted vaccine in comparison to the control group. T cell ELISpot responses peaked at 7-days post boost vaccination with MVA ME-TRAP in all three groups. TRAP-specific IgG responses were highest at 28-days post boost with MVA ME-TRAP in all three groups. There were no differences in cellular and humoral immunogenicity at any of the time points between the control group and the adjuvanted groups. We demonstrate that Matrix-M can be safely used in combination with ChAd63-MVA ME-TRAP heterologous prime-boost immunization without any reduction in cellular or humoral immunogenicity.Clinical Trials Registration NCT01669512.     

Effect of seasonal vaccination on the selection of influenza A/H3N2 epidemic variants

The effect of vaccination on the dynamics of influenza virus variants remains largely unknown in humans, unlike in poultry. In this study, we compared influenza hemagglutinin (HA) gene sequences isolated from vaccinated and unvaccinated populations with the yearly vaccine strains. In total, 181 influenza A/H3N2 virus samples isolated from 82 vaccinated and 99 unvaccinated patients (2011–15, four Japanese influenza seasons) were genetically analyzed using a next-generation sequencer. Amino acid (AA) differences from corresponding vaccine strains were found in 74 of 329 HA1 sites. There was a maximum of four AA differences within the epitopes in the former three seasons (2011–14) and fifteen in the latter season (2014–15). Deviation to a greater number of AA differences was found more significantly in the isolates from vaccinated patients as compared to unvaccinated patients (P = 0.0005 in 2011–14; P = 0.0096 in 2014–15). AA difference rates within epitopes were also significantly higher in the isolates from vaccinated patients than from unvaccinated patients (2.64% vs. 2.14% for 2011–14, P = 0.033; 7.78% vs. 6.59% for 2014–15, P = 0.058). The AA differences at seven sites (48I-278K, 128A-142G, 145S, 158K, and 193S) became dominant in the following seasons. In all of these sites, the dominance was retained during the mismatch of isolates with the vaccine strains and was lost after vaccine match. Our data suggest that in humans, immune pressure induced by vaccination works to select influenza variants genetically distant from vaccine strains.     

Birth outcomes for Australian mother-infant pairs who received an influenza vaccine during pregnancy, 2012–2014: The FluMum study

IntroductionIn Australia, influenza vaccination is recommended for all women who will be pregnant during the influenza season. Vaccine safety and effectiveness are key concerns and influencers of uptake for both vaccine providers and families. We assessed the safety of receiving an influenza vaccination during any trimester of pregnancy with respect to preterm births and infant birthweight.MethodsWe conducted a nested retrospective cohort study of ‘FluMum’ participants (2012–2014). Our primary exposure of interest was influenza vaccination during pregnancy. The primary outcomes of interest were infant birthweight and weeks’ gestation at birth for live singleton infants. Analyses included comparisons of these birth outcomes by vaccination status and trimester of pregnancy an influenza vaccine was given. We calculated means, proportions, and relative risks and performed multivariable logistic regression for potential confounding factors.ResultsIn the 7126 mother-infant pairs enrolled in this study, mean maternal age at infant birth was 31.7 years. Influenza vaccine uptake in pregnancy was 34%. Most mothers with a known date of vaccination received a vaccine in the second trimester (51%). Those mothers with a co-morbidity or risk factor were 13% more likely to have influenza vaccine during pregnancy compared to other mothers (RR 1.13, 95% CI 1.04–1.24, p = 0.007). Mean weeks’ gestation at birth was 38.7 for the vaccinated and 38.8 for the unvaccinated group (p = 0.051). Infants in the vaccinated group weighed 15 g less in birthweight compared to the unvaccinated infants (95% CI −12.8 to 42.2, p = 0.29).ConclusionResults arising from this large Australian cohort study are reassuring with respect to two critical safety outcomes; preterm births and low infant birthweights. Studies examining a broader range of birth outcomes following influenza vaccination during pregnancy are required, particularly now that maternal vaccination in pregnancy has expanded to include pertussis as well as influenza.     

Brief education to promote maternal influenza vaccine uptake: A randomized controlled trial

BackgroundAlthough pregnant women are the highest priority group for seasonal influenza vaccination, maternal influenza vaccination rates remain suboptimal. The purpose of this study was to evaluate the effect of a brief education intervention on maternal influenza vaccine uptake.MethodsDuring the 2013–14 and 2014–15 influenza seasons, we recruited 321 pregnant women from the antenatal clinics of 4 out of 8 public hospitals in Hong Kong with obstetric services. Hospitals were geographically dispersed and provided services to pregnant women with variable socioeconomic backgrounds. Participants were randomized to receive either standard antenatal care or brief one-to-one education. Participants received telephone follow-up at 2 weeks postpartum. The primary study outcome was self-reported receipt of influenza vaccination during pregnancy. The secondary outcomes were the proportion of participants who initiated discussion about influenza vaccination with a health care professional and the proportion of participants who attempted to get vaccinated.ResultsCompared with participants who received standard care, the vaccination rate was higher among participants who received brief education (21.1% vs. 10%; p = 0.006). More participants in the education group initiated discussion about influenza vaccination with their HCP (19.9% vs. 13.1%; p = 0.10), but the difference was not statistically significant. Of participants who did not receive the influenza vaccine (n = 271), 45 attempted to get vaccinated. A significantly higher proportion of participants who attempted to get vaccinated were in the intervention group (82.2% vs. 17.8%; p < 0.001). If participants who had attempted vaccination had received the vaccine, vaccination rates would have been substantially higher (44.1% vs. 15%; p < 0.001). Twenty-six participants were advised against influenza vaccination by a healthcare professional, including general practitioners, obstetricians, and nurses.ConclusionAlthough brief education was effective in improving vaccination uptake among pregnant women, overall vaccination rates remain suboptimal. Multicomponent approaches, including positive vaccination recommendations by healthcare professionals, are needed to promote maternal influenza vaccination.Clinical Trial Registration: www.clinicaltrials.gov (NCT01772901).     

Advax delta inulin adjuvant overcomes immune immaturity in neonatal mice thereby allowing single–dose influenza vaccine protection

Neonates are at high risk for influenza morbidity and mortality due to immune immaturity and lack of priming by prior influenza virus exposure. Inactivated influenza vaccines are ineffective in infants under six months and to provide protection in older children generally require two doses given a month apart. This leaves few options for rapid protection of infants, e.g. during an influenza pandemic. We investigated whether Advax™, a novel polysaccharide adjuvant based on delta inulin microparticles could help overcome neonatal immune hypo-responsiveness. We first tested whether it was possible to use Advax to obtain single-dose vaccine protection of neonatal pups against lethal influenza infection. Inactivated influenza A/H1N1 vaccine (iH1N1) combined with Advax™ adjuvant administered as a single subcutaneous immunization to 7-day-old mouse pups significantly enhanced serum influenza-specific IgM, IgG1, IgG2a and IgG2b levels and was associated with a 3–4 fold increase in the frequency of splenic influenza-specific IgM and IgG antibody secreting cells. Pups immunized with Advax had significantly higher splenocyte influenza-stimulated IFN-γ, IL-2, IL-4, and IL-10 production by CBA and a 3–10 fold higher frequency of IFN-γ, IL-2, IL-4 or IL-17 secreting T cells by ELISPOT. Immunization with iH1N1 + Advax induced robust protection of pups against virus challenge 3 weeks later, whereas pups immunized with iH1N1 antigen alone had no protection. Protection by Advax-adjuvanted iH1N1 was dependent on memory B cells rather than memory T cells, with no protection in neonatal μMT mice that are B-cell deficient. Hence, Advax adjuvant overcame neonatal immune hypo-responsiveness and enabled single-dose protection of pups against otherwise lethal influenza infection, thereby supporting ongoing development of Advax™ as a neonatal vaccine adjuvant.     

Archaeal glycolipid adjuvanted vaccines induce strong influenza-specific immune responses through direct immunization in young and aged mice or through passive maternal immunization

Vaccine induced responses are often weaker in those individuals most susceptible to infection, namely the very young and the elderly, highlighting the need for safe and effective vaccine adjuvants. Herein we evaluated different archaeosome formulations as an adjuvant to the H1N1 influenza hemagglutinin protein and compared immune responses (anti-HA IgG and hemagglutination inhibition assay titers) as well as protection to an influenza A virus (strain A/Puerto Rico/8/1934 H1N1) homologous challenge to those generated using a squalene-based oil-in-water nano-emulsion, AddaVax™ in a murine model. The impact of age (young adult vs aged) on vaccine induced immune responses as well as the protection in pups due to the transfer of maternal antibodies was measured. Overall, we show that archaeal lipid based adjuvants can induce potent anti-HA responses in young and aged mice that can also be passed from vaccinated mothers to pups. Furthermore, young and aged mice immunized with archaeal lipid adjuvants as well as pups from immunized mothers were protected from challenge with influenza. In addition, we show that a simple admixed archaeosome formulation composed of a single sulfated glycolipid namely sulfated lactosylarchaeol (SLA; 6′-sulfate-β-D-Galp-(1,4)-β-D-Glcp-(1,1)-archaeol) can give equal or better protection compared to AddaVax™ or the traditional antigen-encapsulated archaeosome formulations.     

Effects of Repeated Annual Inactivated Influenza Vaccination among Healthcare Personnel on Serum Hemagglutinin Inhibition Antibody Response to A/Perth/16/2009 (H3N2)-like virus during 2010-11

BackgroundRecently, lower estimates of influenza vaccine effectiveness (VE) against A(H3N2) virus illness among those vaccinated during the previous season or multiple seasons have been reported; however, it is unclear whether these effects are due to differences in immunogenicity.MethodsWe performed hemagglutination inhibition antibody (HI) assays on serum collected at preseason, ∼30 days post-vaccination, and postseason from a prospective cohort of healthcare personnel (HCP). Eligible participants had medical and vaccination records for at least four years (since July, 2006), including 578 HCP who received 2010-11 trivalent inactivated influenza vaccine [IIV3, containing A/Perth/16/2009-like A(H3N2)] and 209 HCP who declined vaccination. Estimates of the percentage with high titers (≥40 and > 100) and geometric mean fold change ratios (GMRs) to A/Perth/16/2009-like virus by number of prior vaccinations were adjusted for age, sex, race, education, household size, hospital care responsibilities, and study site.ResultsPost-vaccination GMRs were inversely associated with the number of prior vaccinations, increasing from 2.3 among those with 4 prior vaccinations to 6.2 among HCP with zero prior vaccinations (F[4,567] = 9.97, p < .0005). Thirty-two percent of HCP with 1 prior vaccination achieved titers >100 compared to only 11% of HCP with 4 prior vaccinations (adjusted odds ratio = 6.8, 95% CI = 3.1 – 15.3).ConclusionOur findings point to an exposure-response association between repeated IIV3 vaccination and HI for A(H3N2) and are consistent with recent VE observations. Ultimately, better vaccines and vaccine strategies may be needed in order to optimize immunogenicity and VE for HCP and other repeated vaccinees.     

Comparison of AS03 and Alum on immune responses elicited by A/H3N2 split influenza vaccine in young,mature and aged BALB/c mice

IntroductionThere is great interest in developing more effective influenza vaccines for the elderly. Oil-in-water adjuvants can boost humoral responses to seasonal vaccines in elderly subjects but relatively little is known about their mechanism of action.MethodsWe compared humoral and cellular immune profiles in young adult (2 months), mature (11–12 months) or aged (16–17 month) female BALB/c mice following two doses of Alum or AS03-adjuvanted A/H3N2 split-virus antigen (A/Uruguay/716/2007) at 0.75 or 3 μg hemagglutinin (HA) per dose intramuscularly versus 3 μg HA without adjuvant.ResultsOverall, hemagglutination inhibition (HAI), microneutralization (MN) and end-point ELISA titres were higher in the young mice and when an adjuvant was used. Both adjuvants increased humoral responses in older animals but the highest titres across all groups were observed in the AS03-adjuvanted groups. Neither IgG avidity nor A/H3N2-specific splenocyte proliferation was influenced by age, antigen dose or adjuvant. In contrast, cytokine production by ex vivo-stimulated splenocytes differed widely between groups. Most cytokine levels in older mice vaccinated with antigen alone (3 μg HA/dose) were ≤50% of those in young animals. In young mice, cytokine levels increased modestly with Alum and significantly with AS03. Increases tended to be greatest at the lower antigen dose (0.75 μg versus 3 μg HA). In the older animals, Alum had little impact on cytokine production but responses in the AS03 groups paralleled those of the young mice (broad activation of Th1, Th2, and Th17-type cytokines) and the greatest increases were seen with the higher antigen dose (3 μg HA).ConclusionsIn both young and aged mice, Alum and AS03 increased the magnitude of humoral and cellular responses to split influenza virus vaccination. Overall, these effects were most pronounced in the younger animals and the groups receiving AS03. These data support the use of oil-in-water adjuvants in influenza vaccines targeting the elderly.     

Onjisaponins, from the root of Polygala tenuifolia Willdenow, as effective adjuvants for nasal influenza and diphtheria-pertussis-tetanus vaccines

Active substances from hot water extracts from 267 different Chinese and Japanese medicinal herbs were screened for mucosal adjuvant activity with influenza HA vaccine in mice. The extract from the root of Polygala tenuifolia was found to contain potent mucosal adjuvant activity. The active substances were purified and identified as onjisaponins A, E, F, and G. When each onjisaponin (10 μg) was intranasally (i.n.) inoculated with influenza vaccine (10 μg) in mice, serum hemagglutination-inhibiting (HI) antibody titers increased 3–14 times over control mice administered vaccine alone after 4 weeks. When each onjisaponin (10 μg) was i.n. inoculated with the vaccine (10 μg) followed by i.n. vaccination of the vaccine alone after 3 weeks, serum HI antibody titers increased 27–50 fold over those mice given i.n. vaccinations without onjisaponins. These same conditions also significantly increased nasal anti-influenza virus IgA antibody titers. Two inoculations with onjisaponin F (1 μg) and influenza HA vaccine (1 μg) at 3 weeks intervals, significantly increased serum HI antibody and nasal anti-influenza virus IgA and IgG antibody titers after only 1 week over mice given HA vaccine alone after the secondary vaccination. Intranasal vaccination with onjisaponin F inhibited proliferation of mouse adapted influenza virus A/PR/8/34 in bronchoalveolar lavages of infected mice. Separate intranasal vaccinations with onjisaponins A, E, F, and G (10 μg) each and diphtheria–pertussis–tetanus (DPT) vaccine (10 μg) of mice followed by i.n. vaccination with DPT vaccine alone after 4 weeks showed significant increases in serum IgG and nasal IgA antibody titers after 2 weeks following secondary vaccination over mice vaccinated with DPT vaccine alone. All onjisaponins showed little hemolytic activity at concentrations up to 100 μg/ml. The results of this study suggest that onjisaponins may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines.     

Enhanced humoral response to influenza vaccine in aged mice with a novel adjuvant,rOv-ASP-1

Immunization is the best way to prevent seasonal epidemics and pandemics of influenza. There are two kinds of influenza vaccines available in the United States: an inactivated vaccine (TIV) and an attenuated vaccine; however, only TIV is approved for immunization of the elderly population. While the aged population has the highest rate of influenza vaccination, the protective efficacy is low as evidenced by elderly individuals having the highest mortality associated with influenza. Recently, we reported that an adjuvant derived from the helminth parasite Onchocerca volvulus, named O. volvulus activation-associated secreted protein-1 (Ov-ASP-1), can significantly enhance the protective efficacy of an inactivated vaccine (TIV) in young adult mice. In the current study, we examined whether this recombinant Ov-ASP-1 (rOv-ASP-1) can enhance the efficacy of TIV in aged mice as well. While primary immunization with TIV alone produced only a low level of influenza-specific antibodies (total IgG, IgG1, and IgG2c) in aged mice, the antibody levels were significantly increased after immunization with TIV + rOv-ASP-1. More importantly, the level of the total IgG in aged mice administered TIV + rOv-ASP-1 was comparable to that of young adult mice immunized with TIV alone. Co-administration of rOv-ASP-1 induced a low level of cross-reactive antibody and enhanced the protective efficacy of TIV in aged mice, reflected by significantly increased survival after challenge with a heterologous influenza virus. rOv-ASP-1 was also superior to the conventional adjuvant alum in inducing specific IgG after TIV immunization in aged mice, and in conferring protection after challenge. These results demonstrate that rOv-ASP-1 may serve as a potential adjuvant for influenza vaccine to improve the efficacy of protection in the elderly.     

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.     

Onjisaponins, from the root of Polygala tenuifolia Willdenow, as effective adjuvants for nasal influenza and diphtheria–pertussis–tetanus vaccines

Active substances from hot water extracts from 267 different Chinese and Japanese medicinal herbs were screened for mucosal adjuvant activity with influenza HA vaccine in mice. The extract from the root of Polygala tenuifolia was found to contain potent mucosal adjuvant activity. The active substances were purified and identified as onjisaponins A, E, F, and G. When each onjisaponin (10 μg) was intranasally (i.n.) inoculated with influenza vaccine (10 μg) in mice, serum hemagglutination-inhibiting (HI) antibody titers increased 3–14 times over control mice administered vaccine alone after 4 weeks. When each onjisaponin (10 μg) was i.n. inoculated with the vaccine (10 μg) followed by i.n. vaccination of the vaccine alone after 3 weeks, serum HI antibody titers increased 27–50 fold over those mice given i.n. vaccinations without onjisaponins. These same conditions also significantly increased nasal anti-influenza virus IgA antibody titers. Two inoculations with onjisaponin F (1 μg) and influenza HA vaccine (1 μg) at 3 weeks intervals, significantly increased serum HI antibody and nasal anti-influenza virus IgA and IgG antibody titers after only 1 week over mice given HA vaccine alone after the secondary vaccination. Intranasal vaccination with onjisaponin F inhibited proliferation of mouse adapted influenza virus A/PR/8/34 in bronchoalveolar lavages of infected mice. Separate intranasal vaccinations with onjisaponins A, E, F, and G (10 μg) each and diphtheria–pertussis–tetanus (DPT) vaccine (10 μg) of mice followed by i.n. vaccination with DPT vaccine alone after 4 weeks showed significant increases in serum IgG and nasal IgA antibody titers after 2 weeks following secondary vaccination over mice vaccinated with DPT vaccine alone. All onjisaponins showed little hemolytic activity at concentrations up to 100 μg/ml. The results of this study suggest that onjisaponins may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines.