Aluminum Hydroxide And Aluminum Phosphate Adjuvants Elicit A Different Innate Immune Response.

Aluminum hydroxide (Al(OH)3) and aluminum phosphate (AlPO4) are widely used adjuvants in human vaccines. However, a rationale to choose one or the other is lacking since the differences between molecular mechanisms of action of these adjuvants are unknown. In the current study, we compared the innate immune response induced by both adjuvants in vitro and in vivo. Proteome analysis of human primary monocytes was used to determine the immunological pathways activated by these adjuvants. Subsequently, analysis of immune cells present at the site of injection and proteome analysis of the muscle tissue revealed the differentially regulated processes related to the innate immune response in vivo. Incubation with Al(OH)3 specifically enhanced the activation of antigen processing and presentation pathways in vitro. In vivo experiments showed that only intramuscular (I.M.) immunization with Al(OH)3 attracted neutrophils, while I.M. immunization with AlPO4 attracted monocytes/macrophages to the site of injection. In addition, only I.M. immunization with Al(OH)3 enhanced the process of hemostasis after 96 hours, possibly related to neutrophilic extracellular trap formation. Both adjuvants differentially regulated various immune system-related processes. The results show that Al(OH)3 and AlPO4 act differently on the innate immune system. We speculate that these different regulations affect the interaction with cells, due to the different physicochemical properties of both adjuvants.

An influenza HA stalk reactive polymeric IgA antibody exhibits anti-viral function regulated by binary interaction between HA and the antibody.

IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 (a low affinity anti-HA stalk clone), as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding (Fab paratope-mediated binding to the HA stalk, and IgA Fc glycan-mediated binding to the HA receptor binding site (RBS)) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies.

An Intranasal OMV-Based Vaccine Induces High Mucosal and Systemic Protecting Immunity Against a SARS-CoV-2 Infection.

The development of more effective, accessible, and easy to administer COVID-19 vaccines next to the currently marketed mRNA, viral vector, and whole inactivated virus vaccines is essential to curtailing the SARS-CoV-2 pandemic. A major concern is reduced vaccine-induced immune protection to emerging variants, and therefore booster vaccinations to broaden and strengthen the immune response might be required. Currently, all registered COVID-19 vaccines and the majority of COVID-19 vaccines in development are intramuscularly administered, targeting the induction of systemic immunity. Intranasal vaccines have the capacity to induce local mucosal immunity as well, thereby targeting the primary route of viral entry of SARS-CoV-2 with the potential of blocking transmission. Furthermore, intranasal vaccines offer greater practicality in terms of cost and ease of administration. Currently, only eight out of 112 vaccines in clinical development are administered intranasally. We developed an intranasal COVID-19 subunit vaccine, based on a recombinant, six-proline-stabilized, D614G spike protein (mC-Spike) of SARS-CoV-2 linked via the LPS-binding peptide sequence mCramp (mC) to outer membrane vesicles (OMVs) from Neisseria meningitidis. The spike protein was produced in CHO cells, and after linking to the OMVs, the OMV-mC-Spike vaccine was administered to mice and Syrian hamsters via intranasal or intramuscular prime-boost vaccinations. In all animals that received OMV-mC-Spike, serum-neutralizing antibodies were induced upon vaccination. Importantly, high levels of spike-binding immunoglobulin G (IgG) and A (IgA) antibodies in the nose and lungs were only detected in intranasally vaccinated animals, whereas intramuscular vaccination only induced an IgG response in the serum. Two weeks after their second vaccination, hamsters challenged with SARS-CoV-2 were protected from weight loss and viral replication in the lungs compared to the control groups vaccinated with OMV or spike alone. Histopathology showed no lesions in lungs 7 days after challenge in OMV-mC-Spike-vaccinated hamsters, whereas the control groups did show pathological lesions in the lung. The OMV-mC-Spike candidate vaccine data are very promising and support further development of this novel non-replicating, needle-free, subunit vaccine concept for clinical testing.

Vaccination Prevented Short-Term Memory Loss, but Deteriorated Long-Term Spatial Memory in Alzheimer's Disease Mice, Independent of Amyloid-ß Pathology.

BACKGROUND: Soluble oligomeric amyloid-ß (Aß), rather than Aß plaques, seems to be the culprit in Alzheimer's disease (AD). Accordingly, a new concept vaccine of small cyclic peptide conjugates, selectively targeting oligomeric Aß, has been developed. OBJECTIVE: Study the therapeutic potential of this new vaccine in a mouse model for AD. METHODS: J20 mice, overexpressing human amyloid precursor protein, were validated for an AD-like phenotype. Then, J20 mice were vaccinated at 2, 3, and 4 months of age and AD phenotype was evaluated at 6, 9, and 12 months of age; or at 9, 10, and 11 months with evaluation at 12 months. Effects on Aß pathology were studied by plaque load (immunohistochemistry; 6E10) and antibody titers against Aß (ELISA). AD behavioral phenotype was evaluated by performance in a battery of cognitive tests. RESULTS: J20 mice displayed age-related Aß plaque development and an AD-like behavioral phenotype. A consistent antibody response to the cyclic peptides was, however, not extended to Aß, leaving plaque load unaffected. Nevertheless, immunization at young ages prevented working- and short-term spatial memory loss, but deteriorated long-term spatial learning and memory, at 12 months of age. Immunization at later ages did not affect any measured parameter. CONCLUSION: J20 mice provide a relevant model for AD to study potential anti-Aß treatment. Early vaccination prevented short-term memory loss at later ages, but deteriorated long-term spatial memory, however without affecting Aß pathology. Later vaccination had no effects, but optimal timing may require further investigation.

The Role of Virulence Proteins in Protection Conferred by Bordetella pertussis Outer Membrane Vesicle Vaccines.

The limited protective immunity induced by acellular pertussis vaccines demands development of novel vaccines that induce broader and longer-lived immunity. In this study, we investigated the protective capacity of outer membrane vesicle pertussis vaccines (omvPV) with different antigenic composition in mice to gain insight into which antigens contribute to protection. We showed that total depletion of virulence factors (bvg(-) mode) in omvPV led to diminished protection despite the presence of high antibody levels. Antibody profiling revealed overlap in humoral responses induced by vaccines in bvg(-) and bvg(+) mode, but the potentially protective responses in the bvg(+) vaccine were mainly directed against virulence-associated outer membrane proteins (virOMPs) such as BrkA and Vag8. However, deletion of either BrkA or Vag8 in our outer membrane vesicle vaccines did not affect the level of protection. In addition, the vaccine-induced immunity profile, which encompasses broad antibody and mixed T-helper 1, 2 and 17 responses, was not changed. We conclude that the presence of multiple virOMPs in omvPV is crucial for protection against Bordetella pertussis. This protective immunity does not depend on individual proteins, as their absence or low abundance can be compensated for by other virOMPs.

Intranasal immunization with outer membrane vesicle pertussis vaccine confers broad protection through mucosal IgA and Th17 responses.

A vaccine based on outer membrane vesicles of pertussis (omvPV) is protective in a mouse-challenge model and induces a broad antibody and mixed Th1/Th2/Th17 response against multiple antigens following subcutaneous immunization. However, this route did not result in mucosal immunity and did not prevent nasopharyngeal colonization. In this study, we explored the potential of intranasal immunization with omvPV. Only intranasal immunization induced strong mucosal immune responses that encompasses enhanced pulmonary and nasal IgA antibody levels, mainly directed against Vag8 and LPS. Furthermore, high numbers of IgA- and IgG-producing plasma cells were detected as well as lung-resident IgA memory B-cells. Finally, only intranasal immunization induced pulmonary Th1/Th17-related cytokine responses. The magnitude and type of systemic immunity was comparable between both routes and included high systemic IgG antibody levels, strong IgG-producing plasma cell responses, memory B-cells residing in the spleen and systemic Th1/Th2/Th17-related cytokine responses. Importantly, only intranasal immunization prevented colonization in both the lungs and the nasal cavity. In conclusion, intranasal omvPV immunization induces mucosal IgA and Th17-mediated responses without influencing the systemic immunity profile. These responses resulted in prevention of Bordetella pertussis colonization in the respiratory tract, including the nasal cavity, thereby potentially preventing transmission.

Human immune responses elicited by an intranasal inactivated H5 influenza vaccine.

Intranasally administered influenza vaccines could be more effective than injected vaccines, because intranasal vaccination can induce virus-specific immunoglobulin A (IgA) antibodies in the upper respiratory tract, which is the initial site of infection. In this study, immune responses elicited by an intranasal inactivated vaccine of influenza A(H5N1) virus were evaluated in healthy individuals naive for influenza A(H5N1) virus. Three doses of intranasal inactivated whole-virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy vinyl polymer, had a notable impact on the induction of nasal IgA antibody responses but not on serum IgG antibody responses. The nasal hemagglutinin (HA)-specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA-specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen-specific Th cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine-induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against influenza A(H5N1) viruses and highlights the need to develop novel means to evaluate intranasal vaccine-induced mucosal immune responses.

In silico identification and modification of T cell epitopes in pertussis antigens associated with tolerance.

The resurgence of whooping cough since the introduction of acellular (protein) vaccines has led to a renewed interest in the development of improved pertussis vaccines; Outer Membrane Vesicles (OMVs) carrying pertussis antigens have emerged as viable candidates. An in silico immunogenicity screen was carried out on 49 well-known Bordetella pertussis proteins in order to better understand their potential role toward the efficacy of pertussis OMVs for vaccine design; seven proteins were identified as being good candidates for including in optimized cellular and acellular pertussis vaccines. We then screened these antigens for putative tolerance-inducing sequences, as proteins with reduced tolerogenicity have improved vaccine potency in preclinical models. We used specialized homology tools (JanusMatrix) to identify peptides in the proteins that were cross-reactive with human sequences. Four of the 19 identified cross-reactive peptides were detolerized in silico using a separate tool, OptiMatrix, which disrupted the potential of these peptides to bind to human HLA and murine MHC. Four selected cross-reactive peptides and their detolerized variants were synthesized and their binding to a set of eight common HLA class II alleles was assessed in vitro. Reduced binding affinity to HLA class II was observed for the detolerized variants compared to the wild-type peptides, highlighting the potential of this approach for designing more efficacious pertussis vaccines.

Activation of Human Monocytes by Colloidal Aluminum Salts.

Subunit vaccines often contain colloidal aluminum salt-based adjuvants to activate the innate immune system. These aluminum salts consist of micrometer-sized aggregates. It is well-known that particle size affects the adjuvant effect of particulate adjuvants. In this study, the activation of human monocytes by hexagonal-shaped gibbsite (ø = 210 ± 40 nm) and rod-shaped boehmite (ø = 83 ± 827 nm) was compared with classical aluminum oxyhydroxide adjuvant (alum). To this end, human primary monocytes were cultured in the presence of alum, gibbsite, or boehmite. The transcriptome and proteome of the monocytes were investigated by using quantitative polymerase chain reaction and mass spectrometry. Human monocytic THP-1 cells were used to investigate the effect of the particles on cellular maturation, differentiation, activation, and cytokine secretion, as measured by flow cytometry and enzyme-linked immunosorbent assay. Each particle type resulted in a specific gene expression profile. IL-1ß and IL-6 secretion was significantly upregulated by boehmite and alum. Of the 7 surface markers investigated, only CD80 was significantly upregulated by alum and none by gibbsite or boehmite. Gibbsite hardly activated the monocytes. Boehmite activated human primary monocytes equally to alum, but induced a much milder stress-related response. Therefore, boehmite was identified as a promising adjuvant candidate.

Antibody Specificity Following a Recent Bordetella pertussis Infection in Adolescence Is Correlated With the Pertussis Vaccine Received in Childhood.

Bordetella (B.) pertussis resurgence affects not only the unvaccinated, but also the vaccinated population. Different vaccines are available, however, it is currently unknown whether the type of childhood vaccination has an influence on antibody responses following a B. pertussis infection later in life. Therefore, the study aim was to profile serum antibody responses in young adults with suspected B. pertussis infections, immunized during childhood with either whole-cell (wPV) or monocomponent acellular pertussis (aPV) vaccines. Serum anti-pertussis toxin (PTx) IgG antibody levels served as an indicator for a recent B. pertussis infection. Leftover sera from a diagnostic laboratory from 36 Danish individuals were included and divided into four groups based on immunization background (aPV vs. wPV) and serum anti-PTx IgG levels (- vs. +). Pertussis-specific IgG/IgA antibody levels and antigen specificity were determined by using multiplex immunoassays (MIA), one- and two-dimensional immunoblotting (1 & 2DEWB), and mass spectrometry. Besides enhanced anti-PTx levels, wPV(+) and aPV(+) groups showed increased IgG and IgA levels against pertactin, filamentous hemagglutinin, fimbriae 2/3, and pertussis outer membrane vesicles (OMV). In the wPV(-) and aPV(-) groups, only low levels of anti-OMV antibodies were detected. 1DEWB demonstrated that antibody patterns differed between groups but also between individuals with the same immunization background and anti-PTx levels. 2DWB analysis for serum IgG revealed 133 immunogenic antigens of which 40 were significantly different between groups allowing to differentiate wPV(+) and aPV(+) groups. Similarly, for serum IgA, 7 of 47 immunogenic protein spots were significantly different. This study demonstrated that B. pertussis infection-induced antibody responses were distinct on antigen level between individuals with either wPV or aPV immunization background. Importantly, only 2DEWB and not MIA could detect these differences indicating the potential of this method. Moreover, in individuals immunized with an aPV containing only PTx in childhood, the infection-induced antibody responses were not limited to PTx alone.

Systems vaccinology and big data in the vaccine development chain.

Systems vaccinology has proven a fascinating development in the last decade. Where traditionally vaccine development has been dominated by trial and error, systems vaccinology is a tool that provides novel and comprehensive understanding if properly used. Data sets retrieved from systems-based studies endorse rational design and effective development of safe and efficacious vaccines. In this review we first describe different omics-techniques that form the pillars of systems vaccinology. In the second part, the application of systems vaccinology in the different stages of vaccine development is described. Overall, this review shows that systems vaccinology has become an important tool anywhere in the vaccine development chain.

Development of a thermostable spray dried outer membrane vesicle pertussis vaccine for pulmonary immunization.

Worldwide resurgence of whooping cough calls for improved, next-generation pertussis vaccines that induce broad and long-lasting immunity. A mucosal pertussis vaccine based on outer membrane vesicles (omvPV) is a promising candidate. Further, a vaccine that is stable outside the cold chain would be of substantial advantage for worldwide distribution and application. A vaccine formulated as a powder could both stabilize the vaccine as well as make it suitable for pulmonary vaccination. To that end, we developed a spray dried omvPV with improved stability compared to the liquid omvPV formulation. Spray drying did not affect the structural integrity of the omvPV. The antigenicity of Vag8, a major antigen in omvPV was diminished slightly and an altered tryptophan fluorescence indicated some changes in protein structure. However, when administered via the pulmonary route in mice after reconstitution, spray dried omvPV showed comparable immune responses and protection against challenge with live B. pertussis as liquid omvPV. Mucosal IgA and Th17 responses were established in addition to broad systemic IgG and Th1/Th17 responses, indicating the induction of an effective immunity profile. Overall, a spray dried omvPV was developed that maintained effective immunogenic properties and has an improved storage stability.

Vaccine antigens modulate the innate response of monocytes to Al(OH)3.

Aluminum-based adjuvants have widely been used in human vaccines since 1926. In the absence of antigens, aluminum-based adjuvants can initiate the inflammatory preparedness of innate cells, yet the impact of antigens on this response has not been investigated so far. In this study, we address the modulating effect of vaccine antigens on the monocyte-derived innate response by comparing processes initiated by Al(OH)3 and by Infanrix, an Al(OH)3-adjuvanted trivalent combination vaccine (DTaP), containing diphtheria toxoid (D), tetanus toxoid (T) and acellular pertussis (aP) vaccine antigens. A systems-wide analysis of stimulated monocytes was performed in which full proteome analysis was combined with targeted transcriptome analysis and cytokine analysis. This comprehensive study revealed four major differences in the monocyte response, between plain Al(OH)3 and DTaP stimulation conditions: (I) DTaP increased the anti-inflammatory cytokine IL-10, whereas Al(OH)3 did not; (II) Al(OH)3 increased the gene expression of IFNγ, IL-2 and IL-17a in contrast to the limited induction or even downregulation by DTaP; (III) increased expression of type I interferons-induced proteins was not observed upon DTaP stimulation, but was observed upon Al(OH)3 stimulation; (IV) opposing regulation of protein localization pathways was observed for Al(OH)3 and DTaP stimulation, related to the induction of exocytosis by Al(OH)3 alone. This study highlights that vaccine antigens can antagonize Al(OH)3-induced programming of the innate immune responses at the monocyte level.

Meta-Analysis of Pulmonary Transcriptomes from Differently Primed Mice Identifies Molecular Signatures to Differentiate Immune Responses following Bordetella pertussis Challenge.

Respiratory infection with Bordetella pertussis leads to severe effects in the lungs. The resulting immunity and also immunization with pertussis vaccines protect against disease, but the induced type of immunity and longevity of the response are distinct. In this study the effects of priming, by either vaccination or infection, on a subsequent pathogen encounter were studied. To that end, three postchallenge transcriptome datasets of previously primed mice were combined and compared to the responses in unprimed control mice. In total, 205 genes showed different transcription activity. A coexpression network analysis assembled these genes into 27 clusters, combined into six groups with overlapping biological function. Local pulmonary immunity was only present in mice with infection-induced immunity. Complement-mediated responses were more prominent in mice immunized with an outer membrane vesicle pertussis vaccine than in mice that received a whole-cell pertussis vaccine. Additionally, 46 genes encoding for secreted proteins may serve as markers in blood for the degree of protection (Cxcl9, Gp2, and Pla2g2d), intensity of infection (Retnla, Saa3, Il6, and Il1b), or adaptive recall responses (Ighg, C1qb). The molecular signatures elucidated in this study contribute to better understanding of functional interactions in challenge-induced responses in relation to pertussis immunity.

Bordetella pertussis outer membrane vesicle vaccine confers equal efficacy in mice with milder inflammatory responses compared to a whole-cell vaccine.

The demand for improved pertussis vaccines is urgent due to the resurgence of whooping cough. A deeper understanding of the mode of action of pertussis vaccines is required to achieve this improvement. The vaccine-induced effects of a candidate outer membrane vesicle vaccine (omvPV) and a classical protective but reactogenic whole cell vaccine (wPV) were comprehensively compared in mice. The comparison revealed essential qualitative and quantitative differences with respect to immunogenicity and adverse effects for these vaccines. Both vaccines stimulated a mixed systemic Th1/Th2/Th17 response. Remarkably, omvPV evoked higher IgG levels, lower systemic pro-inflammatory cytokine responses and enhanced splenic gene expression than wPV. The omvPV-induced transcriptome revealed gene signatures of the IFN-signaling pathway, anti-inflammatory signatures that attenuate LPS responses, anti-inflammatory metabolic signatures, and IgG responses. Upon intranasal challenge, both immunized groups were equally efficient in clearing Bordetella pertussis from the lungs. This study importantly shows that immunization with omvPV provides a milder inflammatory responses but with equal protection to bacterial colonization and induction of protective antibody and Th1/Th17 type immune responses compared to wPV. These results emphasize the potential of omvPV as a safe and effective next-generation pertussis vaccine.

Modulating endotoxin activity by combinatorial bioengineering of meningococcal lipopolysaccharide.

Neisseria meningitidis contains a very potent hexa-acylated LPS that is too toxic for therapeutic applications. We used systematic molecular bioengineering of meningococcal LPS through deletion of biosynthetic enzymes in combination with induction of LPS modifying enzymes to yield a variety of novel LPS mutants with changes in both lipid A acylation and phosphorylation. Mass spectrometry was used for detailed compositional determination of the LPS molecular species, and stimulation of immune cells was done to correlate this with endotoxic activity. Removal of phosphethanolamine in lipid A by deletion of lptA slightly reduces activity of hexa-acylated LPS, but this reduction is even more evident in penta-acylated LPS. Surprisingly, expression of PagL deacylase in a penta-acylated lpxL1 mutant increased LPS activity, contradicting the general rule that tetra-acylated LPS is less active than penta-acylated LPS. Further modification included expression of lpxP, an enzyme known to add a secondary 9-hexadecenoic acid to the 2' acyl chain. The LpxP enzyme is temperature-sensitive, enabling control over the ratio of expressed modified hexa- and penta-acylated LPS by simply changing the growth temperature. These LPS derivatives display a broad range of TLR4 activity and differential cytokine induction, which can be exploited for use as vaccine adjuvant or other TLR4-based therapeutics.

Immunological Signatures after Bordetella pertussis Infection Demonstrate Importance of Pulmonary Innate Immune Cells.

Effective immunity against Bordetella pertussis is currently under discussion following the stacking evidence of pertussis resurgence in the vaccinated population. Natural immunity is more effective than vaccine-induced immunity indicating that knowledge on infection-induced responses may contribute to improve vaccination strategies. We applied a systems biology approach comprising microarray, flow cytometry and multiplex immunoassays to unravel the molecular and cellular signatures in unprotected mice and protected mice with infection-induced immunity, around a B. pertussis challenge. Pre-existing systemic memory Th1/Th17 cells, memory B-cells, and mucosal IgA specific for Ptx, Vag8, Fim2/3 were detected in the protected mice 56 days after an experimental infection. In addition, pre-existing high activity and reactivation of pulmonary innate cells such as alveolar macrophages, M-cells and goblet cells was detected. The pro-inflammatory responses in the lungs and serum, and neutrophil recruitment in the spleen upon an infectious challenge of unprotected mice were absent in protected mice. Instead, fast pulmonary immune responses in protected mice led to efficient bacterial clearance and harbored potential new gene markers that contribute to immunity against B. pertussis. These responses comprised of innate makers, such as Clca3, Retlna, Glycam1, Gp2, and Umod, next to adaptive markers, such as CCR6+ B-cells, CCR6+ Th17 cells and CXCR6+ T-cells as demonstrated by transcriptome analysis. In conclusion, besides effective Th1/Th17 and mucosal IgA responses, the primary infection-induced immunity benefits from activation of pulmonary resident innate immune cells, achieved by local pathogen-recognition. These molecular signatures of primary infection-induced immunity provided potential markers to improve vaccine-induced immunity against B. pertussis.

Meningococcal Outer Membrane Vesicle Composition-Dependent Activation of the Innate Immune Response.

Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen-associated molecular patterns, including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an extremely potent hexa-acylated LPS, leading to adverse effects when its OMVs are applied as vaccines. To create safe OMV vaccines, detergent treatment is generally used to reduce the LPS content. While effective, this method also leads to loss of protective antigens such as lipoproteins. Alternatively, genetic modification of LPS can reduce its toxicity. In the present study, we have compared the effects of standard OMV isolation methods using detergent or EDTA with those of genetic modifications of LPS to yield a penta-acylated lipid A (lpxL1 and pagL) on the in vitro induction of innate immune responses. The use of detergent decreased both Toll-like receptor 4 (TLR4) and TLR2 activation by OMVs, while the LPS modifications reduced only TLR4 activation. Mutational removal of PorB or lipoprotein factor H binding protein (fHbp), two proteins known to trigger TLR2 signaling, had no effect, indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent-treated OMVs and lpxL1 OMVs showed similar reductions of cytokine profiles in the human monocytic cell line MM6 and human dendritic cells (DCs). OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß but not of IP-10, a typical TRIF-dependent chemokine. Taken together, these data show that lipid A modification can be used to obtain OMVs with reduced activation of innate immunity, similar to what is found after detergent treatment.

IgG1 Fc N-glycan galactosylation as a biomarker for immune activation.

Immunoglobulin G (IgG) Fc N-glycosylation affects antibody-mediated effector functions and varies with inflammation rooted in both communicable and non-communicable diseases. Worldwide, communicable and non-communicable diseases tend to segregate geographically. Therefore, we studied whether IgG Fc N-glycosylation varies in populations with different environmental exposures in different parts of the world. IgG Fc N-glycosylation was analysed in serum/plasma of 700 school-age children from different communities of Gabon, Ghana, Ecuador, the Netherlands and Germany. IgG1 galactosylation levels were generally higher in more affluent countries and in more urban communities. High IgG1 galactosylation levels correlated with low total IgE levels, low C-reactive protein levels and low prevalence of parasitic infections. Linear mixed modelling showed that only positivity for parasitic infections was a significant predictor of reduced IgG1 galactosylation levels. That IgG1 galactosylation is a predictor of immune activation is supported by the observation that asthmatic children seemed to have reduced IgG1 galactosylation levels as well. This indicates that IgG1 galactosylation levels could be used as a biomarker for immune activation of populations, providing a valuable tool for studies examining the epidemiological transition from communicable to non-communicable diseases.

Immunization with Small Amyloid-ß-derived Cyclopeptide Conjugates Diminishes Amyloid-ß-Induced Neurodegeneration in Mice.

BACKGROUND: Soluble oligomeric (misfolded) species of amyloid-ß (Aß) are the main mediators of toxicity in Alzheimer's disease (AD). These oligomers subsequently form aggregates of insoluble fibrils that precipitate as extracellular and perivascular plaques in the brain. Active immunization against Aß is a promising disease modifying strategy. However, eliciting an immune response against Aß in general may interfere with its biological function and was shown to cause unwanted side-effects. Therefore, we have developed a novel experimental vaccine based on conformational neo-epitopes that are exposed in the misfolded oligomeric Aß, inducing a specific antibody response. OBJECTIVE: Here we investigate the protective effects of the experimental vaccine against oligomeric Aß1-42-induced neuronal fiber loss in vivo. METHODS: C57BL/6 mice were immunized or mock-immunized. Antibody responses were measured by enzyme-linked immunosorbent assay. Next, mice received a stereotactic injection of oligomeric Aß1-42 into the nucleus basalis of Meynert (NBM) on one side of the brain (lesion side), and scrambled Aß1-42 peptide in the contralateral NBM (control side). The densities of choline acetyltransferase-stained cholinergic fibers origination from the NBM were measured in the parietal neocortex postmortem. The percentage of fiber loss in the lesion side was determined relative to the control side of the brain. RESULTS: Immunized responders (79%) showed 23% less cholinergic fiber loss (p = 0.01) relative to mock-immunized mice. Moreover, fiber loss in immunized responders correlated negatively with the measured antibody responses (R2 = 0.29, p = 0.02). CONCLUSION: These results may provide a lead towards a (prophylactic) vaccine to prevent or at least attenuate (early onset) AD symptoms.