Outer membrane vesicles obtained from Bordetella pertussis Tohama expressing the lipid A deacylase PagL as a novel acellular vaccine candidate

In an effort to devise a safer and effective pertussis acelullar vaccine, outer membrane vesicles (OMVs) were engineered to decrease their endotoxicity. The pagL gene from Bordetella bronchiseptica, which encodes a lipid A 3-deacylase, was expressed in Bordetella pertussis strain Tohama I. The resulting OMVs, designated OMVs_BpPagL, contain tetra- instead of penta-acylated LOS, in addition to pertussis surface immunogens such as pertactin and pertussis toxin, as the wild type OMVs. The characterized pertussis OMVs_BpPagL were used in murine B. pertussis intranasal (i.n.) challenge model to examine their protective capacity when delivered by i.n. routes. Immunized BALB/c mice were challenged with sublethal doses of B. pertussis. Significant differences between immunized animals and the PBS treated group were observed (p < 0.001). Adequate elimination rates (p < 0.005) were observed in mice immunized either with OMVs_BpPagL and wild type OMVs. All OMV preparations tested were non toxic according to WHO criteria; however, OMVs_BpPagL displayed almost no weight loss at 3 days post administration, indicating less toxicity when compared with wild type OMVs. Induction of IL6- and IL1-expression in lung after i.n. delivery as well as neutrophil recruitment to airways showed coincident results, with a lower induction of the proinflammatory cytokines and lower recruitment in the case of OMVs_BpPagL compared to wild type OMVs.Given their lower endotoxic activity and retained protective capacity in the mouse model, OMVs_BpPagL obtained from B. pertussis seem as interesting candidates to be considered for the development of novel multi-antigen vaccine.     

Nontoxic outer membrane vesicles efficiently increase the efficacy of an influenza vaccine in mice and ferrets

In this study, we developed a further-modified outer membrane vesicle (fmOMV) from the ΔmsbB/ΔpagP mutant of Escherichia coli transformed with the plasmid, pLpxF, in order to use it as an adjuvant for pandemic H1N1 (pH1N1) influenza vaccine. We evaluated the efficacy of the pH1N1 influenza vaccine containing the fmOMV in animal models as compared to the commercial adjuvants, alum or AddaVax^TM. The fmOMV–adjuvanted pH1N1 influenza vaccine induced a significant increase in the humoral immunity; however, this effect was less than that of the AddaVax^TM. The fmOMV–adjuvanted vaccine displayed pronounced an enhanced protective efficacy with increased T cell immune response and reduced the viral load in the lungs of the infected mice after challenging them with a lethal dose of the homologous virus. Moreover, it resulted in a significantly higher cross-protection against heterologous virus challenge than that of the pH1N1 vaccine with alum or with no adjuvants. In ferrets, the fmOMV–adjuvanted vaccine elicited a superior antibody response based on the HI titer and efficiently protected the animals from the lethal viral challenges. Taken together, the nontoxic fmOMV could be a promising adjuvant for inducing robust T cell priming into the pH1N1 vaccine and might be broadly applicable to the development of preventive measures against influenza virus infection.     

Mucosal immunization with Shigella flexneri outer membrane vesicles induced protection in mice

Vaccination appears to be the only rational prophylactic approach to control shigellosis. Unfortunately, there is still no safe and efficacious vaccine available. We investigated the protection conferred by a new vaccine containing outer membrane vesicles (OMVs) from Shigella flexneri with an adjuvant based on nanoparticles in an experimental model of shigellosis in mice. OMVs were encapsulated in poly(anhydride) nanoparticles prepared by a solvent displacement method with the copolymer PMV/MA. OMVs loaded into NPs (NP-OMVs) were homogeneous and spherical in shape, with a size of 197 nm (PdI = 0.06). BALB/c mice (females, 9-week-old, 20 ± 1 g) were immunized by intradermal, nasal, ocular (20 μg) or oral route (100 μg) with free or encapsulated OMV. Thirty-five days after administration, mice were infected intranasally with a lethal dose of S. flexneri (1 × 10⁷ CFU). The new vaccine was able to protect fully against infection when it was administered via mucosa. By intradermal route the NP-OMVs formulation increased the protection from 20%, obtained with free extract, to 100%. Interestingly, both OMVs and OMV-NP induced full protection when administered by the nasal and conjuntival route. A strong association between the ratio of IL-12p40/IL-10 and protection was found. Moreover, low levels of IFN-γ correlate with protection. Under the experimental conditions used, the adjuvant did not induce any adverse effects. These results place OMVs among promising candidates to be used for vaccination against Shigellosis.     

Development of a cost-effective vaccine candidate with outer membrane vesicles of a tolA-disrupted Shigella boydii strain

Our previous studies on outer membrane vesicles based vaccine development against shigellosis, revealed the inability of Shigella to release significant amount of vesicles naturally, during growth. Disruption of tolA, one of the genes of the Tol–Pal system of Gram negative bacterial membrane, has increased the vesicle release rate of a Shigella boydii type 4 strain to approximately 60% higher. We also noticed the vesicles, released from tolA-disrupted strain captured more OmpA protein and lipopolysaccharide, compared to the vesicles released from its wild type prototype. Six to seven weeks old BALB/c mice, immunized with 25 μg of three oral doses of the vesicles, released by tolA mutant, conferred 100% protection against lethal homologous challenge through nasal route, compared to only 60% protection after the same dose of wild type immunogen. Mice, immunized with the vesicles from tolA-mutant, manifested significant secretion of mucosal IgG and IgA. A sharp and significant response of pro-inflammatory cytokines (TNF-α, IL-6, IFN-γ) were also observed in the lung lavage of these groups of mice, within 6 h post challenge; but at 24 h, these inflammatory cytokines showed the sign of subsidence and the system was taken over by the release of anti-inflammatory cytokines (IL-4 and IL-10). Studies with naïve peritoneal macrophages, proved further, the potency of these vesicles to stimulate nitric oxide and TNF-α, IL-12p70, IL-6 and IL-10 productions in-vitro. The ability of these vesicles to trigger polarization of CD4⁺ T cells toward Th1 adaptive immune response, had also been observed along with the presence of anti-inflammatory cytokines in the system. Our study demonstrated, the vesicles from tolA-disrupted Shigella were able to suppress Shigella-mediated inflammation in the host and could balance between inflammation and anti-inflammation, promoting better survival and health of the infected mice. Outer membrane vesicles from tolA-mutant, could be a potential cost-effective vaccine candidate against shigellosis.     

Outer membrane vesicles (OMV) production of Neisseria meningitidis serogroup B in batch process

Serogroup B outer membrane vesicles (OMV) with iron regulated proteins (IRP) from Neisseria meningitidis constitute the antigen for the vaccine against the disease caused by this bacterium. Aiming to enhance final OMV concentration, seven batch experiments were carried out under four different conditions: (i) with original Catlin medium; (ii) with original Catlin medium and lactate and amino acids pulse at the 6th cultivation hour; (iii) with Catlin medium with double initial concentrations of lactate and amino acids and (iv) Catlin medium without glycerol and with double initial concentrations of lactate and amino acids. The cultivation experiments were carried out in a 7-L bioreactor under the following conditions: 36°C, 0.5atm, overlay air 1L/min, agitation: 250-850rpm, and O(2) control at 10%, 20h. After lactate and amino acids exhaustion, cell growth reached stationary phase and a significant release increase of OMV was observed. According to the Luedeking & Piret model, OMV liberation is non-growth associated. Glycerol was not consumed during cultivation. The maximum OMV concentration value attained was 162mg/L with correspondent productivity of 8.1mg/(Lh) employing Catlin medium with double initial concentrations of lactate and amino acids. The obtained OMV satisfied constitution and protein pattern criteria and were suitable for vaccine production.     

Bivalent non-typhoidal Salmonella outer membrane vesicles immunized mice sera confer passive protection against gastroenteritis in a suckling mice model

Invasive non-typhoidal Salmonella (iNTS) serovars, especially Salmonella Typhimurium (ST) and Salmonella Enteritidis (SE), cause gastroenteritis worldwide. Due to the emergence of multi-drug resistance in iNTS, a broad-spectrum vaccine is urgently needed for the prevention of iNTS infection. Currently, there is no effective licensed vaccine against iNTS available in the market. We have formulated an outer membrane vesicles (OMVs) based bivalent immunogen as a vaccine candidate to generate broad-spectrum protective immunity against both recently circulating prevalent ST and SE. We have isolated OMVs from ST and SE and formulated the immunogen by mixing both OMVs (1:1 ratio). Three doses of bivalent immunogen significantly induced humoral immune responses against lipopolysaccharides (LPSs) and outer membrane proteins (OMPs) as well as a cell-mediated immune response in adult mice. We also observed that proteins of OMVs act as an adjuvant for generation of high levels of anti-LPS antibodies through T cell activation. We then characterized the one-day old suckling mice model for both ST and SE mediated gastroenteritis and used the model for a passive protection study. In the passive protection study, we found the passive transfer of bivalent OMVs immunized sera significantly reduced ST and SE mediated colonization and gastroenteritis symptoms in the colon of suckling mice compared to non-immunized sera recipients. The overall study demonstrated that OMVs based bivalent vaccine could generate broad-spectrum immunity against prevalent iNTS mediated gastroenteritis. This study also established the suckling mice model as a suitable animal model for vaccine study against iNTS mediated gastroenteritis.     

Increased immunogenicity of the MF59-adjuvanted influenza vaccine compared to a conventional subunit vaccine in elderly subjects

Three-hundred and eight outpatient elderly subjects ( 65 years) were randomly assigned to receive the MF59-adjuvanted influenza vaccine (FLUAD; n = 204) or a conventional subunit influenza vaccine (AGRIPPAL S1; n = 104) in order to compare the safety and immunogenicity of the two vaccines. Although mild pain at the injection site was reported more frequently by subjects immunised with the adjuvanted vaccine, both vaccines were shown to be safe and well tolerated. The adjuvanted vaccine was more immunogenic as indicated by higher post-immunisation geometric mean titres (GMTs) and by higher proportions of subjects with post-immunisation four fold increases of antibody titres or subjects with 1/160 post-immunisation HI titres. These differences, statistically significant for all three strains after immunisation, indicated that, by addition of the MF59 adjuvant emulsion, conventional subunit influenza antigens acquire an enhanced immunogenicity without any clinically significant increase of their reactogenicity.     

Safety and immunogenicity of MF59-adjuvanted influenza vaccine in the elderly

Safety and immunogenicity of the influenza vaccine adjuvanted with MF59 (FLUAD) were compared to those of a non adjuvanted subunit vaccine in elderly subjects during three consecutive influenza seasons. Geometric mean titres and proportions of subjects with either a > or = four-fold increase in antibody titres or with an HI titre > or = 128 after immunisation were higher in FLUAD recipients. The adjuvant effect on the magnitude of the responses was most pronounced in subjects with pre-vaccination titres < or = 40. Although associated with more frequent mild local reactions, the adjuvanted vaccine was well tolerated. Thus, the addition of MF59 increased the immunogenicity of the subunit influenza vaccine in elderly persons with low pre-vaccination titres, who are at greatest risk of developing severe influenza disease and vaccine failure, without a clinically important increase in reactogenicity.     

Efficacy and safety of an intranasal virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A in mice and cotton rats

Respiratory syncytial virus infection remains a serious health problem, not only in infants but also in immunocompromised adults and the elderly. An effective and safe vaccine is not available due to several obstacles: non-replicating RSV vaccines may prime for excess Th2-type responses and enhanced respiratory disease (ERD) upon natural RSV infection of vaccine recipients. We previously found that inclusion of the Toll-like receptor 4 (TLR4) ligand monophosphoryl lipid A (MPLA) in reconstituted RSV membranes (virosomes) potentiates vaccine-induced immunity and skews immune responses toward a Th1-phenotype, without priming for ERD. As mucosal immunization is an attractive approach for induction of RSV-specific systemic and mucosal antibody responses and TLR ligands could potentiate such responses, we explored the efficacy and safety of RSV-MPLA virosomes administered intranasally (IN) to mice and cotton rats. In mice, we found that incorporation of MPLA in IN-administered RSV virosomes increased both systemic IgG and local secretory-IgA (S-IgA) antibody levels and resulted in significantly reduced lung viral titers upon live virus challenge. Also, RSV MPLA virosomes induced more Th1–skewed responses compared to responses induced by FI-RSV. Antibody responses and Th1/Th2-cytokine responses induced by RSV-MPLA virosomes were comparable to those induced by live RSV infection. By comparison, formalin-inactivated RSV (FI-RSV) induced serum IgG that inhibited viral shedding upon challenge, but also induced Th2-skewed responses. In cotton rats, similar effects of incorporation of MPLA in virosomes were observed with respect to induction of systemic antibodies and inhibition of lung viral shedding upon challenge, but mucosal sS-IgA responses were only moderately enhanced. Importantly, IN immunization with RSV-MPLA virosomes, like live virus infection, did not lead to any signs of ERD upon live virus challenge of vaccinated animals, whereas IM immunization with FI-RSV did induce severe lung immunopathology under otherwise comparable conditions. Taken together, these data show that mucosally administered RSV-MPLA virosomes hold promise for a safe and effective vaccine against RSV.     

Construction of an inhalable recombinant M2e-FP-expressing Bacillus subtilis spores-based vaccine and evaluation of its protection efficacy against influenza in a mouse model

Influenza A virus (IAV) is a deadly zoonotic pathogen that remains a burden to global health systems despite continuous vaccinations, indicating the need for an improved vaccine strategy. In this work, we constructed a new recombinant influenza vaccine using Bacillus subtilis spores expressing M2e-FP protein (RSM2eFP) and assessed its potency and efficacy in BALB/c mouse immunized via aerosolized intratracheal inoculation (i.t.) or intragastric (i.g.) administration. Immunization via i.t. route conferred 100 % protection against 20 × LD₅₀ A/PR/8/34 (H1N1) virus compared with only 50 % via the i.g. route. Even when challenged with 40 × LD₅₀ virus, the RSM2eFP vaccine immunized via i.t. provided 80 % protection. Consistently, i.t. inoculation of RSM2eFP spore vaccine induced a stronger lung mucosal immune response and a greater cellular immune response than i.g. administration, as indicated by the high production of IgG and SIgA. In addition, the RSM2eFP spore vaccine diminished the yield of infectious virus in the lung of mice immunized via i.t. These results suggest that i.t. immunization of the RSM2eFP spore vaccine may be a promising strategy for the development of mucosal vaccines against IAV infections.     

A single dose and long lasting vaccine against pandemic influenza through the controlled release of a heterospecies tandem M2 sequence embedded within detoxified bacterial outer membrane vesicles

The influenza A virus undergoes genetic drift and shift, leaving the general population susceptible to emerging pandemic strains, despite seasonal flu vaccination. Here we describe a single dose influenza vaccine derived from recombinant outer membrane vesicles (rOMVs) that display an antigen-mapped heterospecies tandem sequence of the M2 protein from the influenza A virus, released over 30 days from poly(lactic-co-glycolide) (PLGA) microparticles. Four weeks post vaccination, BALB/c mice developed high anti-M2e IgG titers that were equivalent to those generated at 8 weeks in a typical prime/boost vaccine regimen. Challenge of mice with a lethal dose of mouse adapted influenza virus PR8 (H1N1) 10 weeks post vaccination resulted in 100% survival for both rOMV single-dose microparticle and prime/boost vaccinated mice. Anti-M2e IgG1 and IgG2a antibody titers were weighted toward IgG1, but splenocytes isolated from rOMV single-dose microparticle vaccinated mice produced high levels of IFNγ relative to IL-4 in response to stimulation with M2e peptides, supporting a more Th1 biased immune response. The protective immune response was long lasting, eliciting sustained antibody titers and 100% survival of mice challenged with a lethal dose of PR8 six months post initial vaccination. Together, these data support the potential of controlled release rOMVs as an effective single dose, long lasting and rapidly effective vaccine to protect against influenza.     

Stable emulsion (SE) alone is an effective adjuvant for a recombinant,baculovirus-expressed H5 influenza vaccine in healthy adults: A Phase 2 trial

BackgroundInfluenza A viruses of the H5 subtype have been identified as important targets for development of vaccines. Achievement of potentially protective antibody responses against pandemic strains has usually required the use of adjuvants.ObjectivesWe evaluated a candidate A/Indonesia/05/2005 (H5) vaccine generated by baculovirus expression of recombinant hemagglutinin (HA) protein with or without stable emulsion (SE) as an adjuvant.MethodsHealthy subjects 18–49 years old were randomized (1:1:1:1) to receive two doses of rHA at 7.5 ug per dose (no adjuvant), or 3.8 ug, 7.5 ug, or 15 ug per dose formulated with 2% SE separated by 21 days, and serum from day 0, 21, 42, and 201 assessed by hemagglutination-inhibition.Results341 subjects were enrolled in the study and 321 received two doses of vaccine. Vaccination was well tolerated in all groups. After two doses, seroconversion was noted in only 9% (95% confidence interval 4%, 17%) of recipients of unadjuvanted vaccine at 7.5 ug, but in 70% (59%, 80%), 76% (65%, 85%), and 83% (73%, 91%) of those receiving adjuvanted vaccine at 3.8 ug, 7.5 ug, or 15 ug respectively.ConclusionsStable emulsion alone is an effective adjuvant for rH5 vaccine in healthy adults. All three adjuvanted dose groups met the current criterion for seroconversion rate for pandemic vaccines. This dose-ranging study also identified a group (15 ug per dose formulated with 2% SE) that met the criteria for both seroconversion and percentage of subjects achieving an HI antibody titer ⩾ 40. These Phase 2 data support the further clinical development of SE adjuvanted Panblok H5.Clinical trial registration: NCT01612000.The protocol was approved by the relevant Institutional Review Board for each study site, and the study was conducted in accordance with the Declaration of Helsinki, International Conference of Harmonisation – Good Clinical Practice, and all applicable laws and regulations. All participants provided written informed consent before study procedures.     

A randomized,double-blind,controlled clinical trial to evaluate the safety and immunogenicity of an intranasally administered trivalent inactivated influenza vaccine with adjuvant LTh(αK): A phase I study

Background

A nasal influenza vaccine has been available only in a live attenuated form, which limits the range of recipients to immune-competent individuals. The present study evaluated a newly developed intranasal inactivated influenza vaccine with a novel adjuvant, heat-labile enterotoxin (LT) derived from E. coli (LTh(αK)).

Improving the immunogenicity of a trivalent Neisseria meningitidis native outer membrane vesicle vaccine by genetic modification

Trivalent native outer membrane vesicles (nOMVs) derived from three genetically modified Neisseria meningitidis serogroup B strains have been previously evaluated immunologically in mice and rabbits. This nOMV vaccine elicited serum bactericidal activity (SBA) against multiple N. meningitidis serogroup B strains as well as strains from serogroups C, Y, W, and X. In this study, we used trivalent nOMVs isolated from the same vaccine strains and evaluated their immunogenicity in an infant Rhesus macaque (IRM) model whose immune responses to the vaccine are likely to be more predictive of the responses in human infants. IRMs were immunized with trivalent nOMV vaccines and sera were evaluated for exogenous human serum complement-dependent SBA (hSBA). Antibody responses to selected hSBA generating antigens contained within the trivalent nOMVs were also measured and we found that antibody titers against factor H binding protein variant 2 (fHbpv2) were very low in the sera from animals immunized with these original nOMV vaccines. To increase the fHbp content in the nOMVs, the vaccine strains were further genetically altered by addition of another fHbp gene copy into the porB locus. Trivalent nOMVs from the three new vaccine strains had higher fHbp antigen levels and generated higher anti-fHbp antibody responses in immunized mice and IRMs. As expected, fHbp insertion into the porB locus resulted in no PorB expression. Interestingly, higher expression of PorA, an hSBA generating antigen, was observed for all three modified vaccine strains. Compared to the trivalent nOMVs from the original strains, higher PorA levels in the improved nOMVs resulted in higher anti-PorA antibody responses in mice and IRMs. In addition, hSBA titers against other strains with PorA as the only hSBA antigen in common with the vaccine strains also increased.     

Frequencies of peripheral immune cells in older adults following seasonal influenza vaccination with an adjuvanted vaccine

As age increases, immune responses and consequently protection following vaccination to seasonal influenza is commonly believed to decrease. Possible drivers of this immune dysfunction include immunosenescence, repeated exposure to the same seasonal influenza antigens, and prior infection with cytomegalovirus (CMV). Here, to determine immune parameters distinguishing vaccine humoral responders (R) from non-responders (NR) following vaccination, we surveyed broad peripheral blood “cellular immune correlates” of older adults vaccinated with Fluad® (an adjuvanted subunit influenza vaccine containing strains H1N1, H3N2 and B). Phenotyping included αβ-T-cells, γδ-T-cells, B-cells and myeloid cells. The frequencies of most of these lymphocyte phenotypes were found to be similar in R and NR, although perhaps counterintuitively, one of the few differences seen between the two groups was higher frequencies of regulatory T-cells in R. These differences were more prominent for responses to the vaccine strains H1N1 and H3N2 than to the B strain, and in CMV-seropositive than CMV-seronegative elderly. Further, frequencies of early-differentiated CD4+ T-cells tended to be higher and frequencies of memory CD4+ T-cells tended to be lower in R than NR. There were also differences in B-cells, with higher frequencies in R compared to NR. To the best of our knowledge, these results are the first to report such differences in elderly people responding or failing to respond to adjuvanted seasonal influenza vaccination.     

Strong local and systemic protective immunity induced in the ferret model by an intranasal virosome-formulated influenza subunit vaccine

The proliferation of influenza viruses causes costly, recurrent, annual epidemics. Current vaccines, mainly administered parenterally, have been shown to be suboptimal in terms of efficacy, particularly where local IgA responses are concerned. Recent investigations of virosomes as delivery systems for viral HA and NA antigens have demonstrated an improved immune response. This paper investigates the efficacy of a novel virosome-based intranasal influenza vaccine by its ability to reduce disease symptoms and its effect on viral shedding in nasal secretions of immunised ferrets. The use of ferrets in the study of influenza vaccines is based on the good comparability between ferret and human response to the disease. Intranasal, as opposed to parenteral, administration of a trivalent virosome-based subunit vaccine adjuvanted with HLT provides an almost total prevention of virus shedding combined with a high level of immunological protection against homologous virus challenge. The ease of application of an intranasal vaccine may have positive repercussions in the adoption of influenza vaccinations, particularly in 'at-risk' groups.     

Dose ranging of adjuvant and antigen in a cell culture H5N1 influenza vaccine: Safety and immunogenicity of a phase 1/2 clinical trial

Background

Dose-sparing strategies and new production technologies will be necessary to produce adequate supplies of vaccines for pandemic influenza. One approach is to include adjuvant, which can reduce the amount of antigen required for immunization and stimulate cross-reactive responses to drifted variants of novel viruses. Dose-sparing studies of adjuvant, itself a finite resource, have not previously been reported for H5N1 vaccine development.

Methodology/principal findings

A total of 753 healthy 18–40-year-old adults were randomized to one of 12 groups (N ∼ 60/group) to receive two intramuscular doses, 21 days apart, of 3.75, 7.5 or 15 μg of cell culture grown influenza A/H5N1 hemagglutinin (A/Indonesia/5/2005 (H5N1)/PR-8-IBCDC-RG2), each dose level formulated with 0%, 25%, 50% or 100% of the MF59 dose contained in licensed influenza vaccine. 752 subjects actually received one dose, and 695 a second dose. Serum hemagglutination inhibition and neutralizing antibody levels, were determined before and 21 days after each dose. Safety and reactogenicity were assessed by self-completed diary cards. Nonadjuvanted H5N1 formulations were poorly immunogenic, but antibody responses were significantly enhanced by all doses of MF59 for each antigen level. The 3.75 μg H5N1 containing 50% MF59 satisfied the European criteria for pandemic vaccine licensure. All formulations were well tolerated, although MF59 dose-dependent increases in the frequency of injection site pain were observed. The frequencies of injection site and systemic reactions were lower after receipt of the second dose of vaccine. No vaccine-related SAE was reported.

Conclusions

Dose-sparing of both antigen and adjuvant is possible without compromising immunogenicity, while improving reactogenicity and is a promising strategy that will expand the availability of vaccines for global control of pandemic influenza.     

Adjuvanting an inactivated influenza vaccine with flagellin improves the function and quantity of the long-term antibody response in a nonhuman primate neonate model

Young infants are at significantly increased risk of developing severe disease following infection with influenza virus. At present there is no approved vaccine for individuals below the age of six months given previous studies showing a failure of these individuals to efficiently seroconvert. Given the major impact of influenza on infant health, it is critical that we develop vaccines that will be safe and effective in this population. Using a nonhuman primate (NHP) model, we have evaluated the ability of an inactivated influenza virus vaccine adjuvanted with flagellin to result in long term immune responses in neonates. To evaluate this critical attribute, neonate NHP were vaccinated and boosted with inactivated influenza virus in combination with either flagellin or a mutant inactive flagellin control. Our studies show that inclusion of flagellin resulted in a significant increase (5-fold, p = 0.04) in influenza virus-specific IgG antibody at 6 months post-vaccination. In addition, the antibody present at this late time was of higher affinity (2.4-fold, p = 0.02). Finally a greater percentage of infants had detectable neutralizing antibody. These results support the use of flagellin in neonates as an adjuvant that promotes long-lived, high affinity antibody responses.     

The effect of mucoadhesive excipient on the nasal retention time of and the antibody responses induced by an intranasal influenza vaccine

IntroductionRecently, we reported that intranasal vaccination of humans with whole inactivated influenza vaccine in the absence of mucosal adjuvant induced neutralizing antibody responses in the serum and nasal mucus. The mucoadhesive excipient carboxy-vinyl polymer (CVP) increases the viscosity and therefore mucoadhesiveness of intranasal medicaments and is an authorized excipient in Japan. In the present study, we analyzed the effect of adding CVP on intranasal whole inactivated influenza vaccine antigen dynamics and antibody responses.MethodsMice and nonhuman primates (NHPs) were intranasally administered the [¹⁸F]-radiolabeled vaccine and subjected to positron emission tomography analysis for 6 h. Dendritic cells were stimulated in vitro with the vaccine mixed with or without a mucosal adjuvant (Ampligen) and/or CVP, after which the tumor necrosis factor (TNF)-α and interferon (IFN)-β levels in the supernatants were measured. Cynomolgus monkeys were immunized intranasally with the vaccine mixed with Ampligen and/or CVP and their vaccine-specific serum IgG and IgA titers were measured on days 0 and 33.ResultsThe vaccine was retained significantly longer in the nasal cavity of both mice and NHPs when it was delivered with CVP rather than PBS. Accumulation of the radiolabeled vaccine in the central nervous system was not detected in either model regardless of whether CVP was used. CVP only very weakly increased the TNF-α production of vaccine-stimulated dendritic cells. IFN-β production was not observed regardless of the presence or absence of CVP. CVP increased the vaccine-specific IgA antibody responses of the intranasally vaccinated cynomolgus macaques.ConclusionCVP increased intranasal retention of whole inactivated influenza vaccine, did not promote antigen redirection to the central nervous system, and improved mucosal antibody responses. The mechanism probably relates to its mucoadhesive properties rather than its ability to directly stimulate the immune system. Intranasal vaccines with CVP may be a promising candidate vaccine formulation for humans.