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Development of new vaccines tailored for difficult-to-target diseases is hampered by a lack of diverse adjuvants for human use, and none of the currently available adjuvants induce Th17 cells. Here, we develop a liposomal adjuvant, CAF®10b, that incorporates Mincle and Toll-like receptor 9 agonists. In parallel mouse and non-human primate studies comparing to CAF® adjuvants already in clinical trials, we report species-specific effects of adjuvant composition on the quality and magnitude of the responses. When combined with antigen, CAF®10b induces Th1 and Th17 responses and protection against a pulmonary infection with Mycobacterium tuberculosis in mice. In non-human primates, CAF®10b induces higher Th1 responses and robust Th17 responses detectable after six months, and systemic and pulmonary Th1 and Th17 recall responses, in a sterile model of local recall. Overall, CAF®10b drives robust memory antibody, Th1 and Th17 vaccine-responses via a non-mucosal immunization route across both rodent and primate species., (© 2024. The Author(s).)

Rapidly waning immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires continued global access to affordable vaccines. Globally, inactivated SARS-CoV-2 vaccines have been widely used during the SARS-CoV-2 pandemic. In this proof-of-concept study we adapted an original-D614G SARS-CoV-2 virus to Vero cell culture as a strategy to enhance inactivated vaccine manufacturing productivity. A passage 60 (P60) virus showed enhanced fitness and 50-fold increased virus yield in a bioreactor compared to the original-D614G virus. It further remained susceptible to neutralization by plasma from SARS-CoV-2 vaccinated and convalescent individuals, suggesting exposure of relevant epitopes. Monovalent inactivated P60 and bivalent inactivated P60/omicron BA.1 vaccines induced neutralizing responses against original-D614G and BA.1 viruses in mice and hamsters, demonstrating that the P60 virus is a suitable vaccine antigen. Antibodies further cross-neutralized delta and BA.5 viruses. Importantly, the inactivated P60 vaccine protected hamsters against disease upon challenge with original-D614G or BA.1 virus, with minimal lung pathology and lower virus loads in the upper and lower airways. Antigenicity of the P60 virus was thus retained compared to the original virus despite the acquisition of cell culture adaptive mutations. Consequently, cell culture adaptation may be a useful approach to increase yields in inactivated vaccine antigen production., (© 2024. The Author(s).)

Vaccine adjuvants, such as liposome-based cationic adjuvant formulations (CAFs), are able to boost immune responses and, by incorporation of distinct immunomodulators, steer immunity towards a desired direction in mice, non-human primates and humans, while less studied in pigs. Here we used commercial pigs to investigate polarizing adjuvant effects of CAFs with immunomodulators: C-type lectin receptor ligands trehalose-6,6'-dibehenate and monomycolyl glycerol, toll-like receptor 3 ligand Poly(I:C) or retinoic acid. Vaccines were formulated with a recombinant Chlamydia model protein antigen and administered via three injection routes. All adjuvants significantly increased antigen-specific IgG in serum, compared to non-adjuvanted antigen. Administering the vaccines through intramuscular and intraperitoneal routes induced significantly higher antigen-specific IgG and IgA serum antibodies, than the perirectal route. Although immunizations triggered cell-mediated immunity, no significant differences between adjuvants or injection sites were detected. Genes depicting T cell subtypes revealed only minor differences. Our findings suggest that specific signatures of the tested adjuvant immunomodulation do not translate well from mice to pigs in standard two-dose immunizations. This study provides new insights into immune responses to CAFs in pigs, and highlights that adjuvant development should ideally be carried out in the intended species of interest or in models with high predictive validity/translational value., Competing Interests: Declaration of competing interest EŠ, GE, JTJ, GJ and GKP are employed by Statens Serum Institut, which is a non-profit government research facility, holding patents on the cationic adjuvant formulations (CAF®)., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

In many autoimmune diseases, autoantigen-specific Th17 cells play a pivotal role in disease pathogenesis. Th17 cells can transdifferentiate into other T cell subsets in inflammatory conditions, however, there have been no attempts to target Th17 cell plasticity using vaccines. We investigated if autoantigen-specific Th17 cells could be specifically targeted using a therapeutic vaccine approach, where antigen was formulated in all-trans retinoic acid (ATRA)-containing liposomes, permitting co-delivery of antigen and ATRA to the same target cell. Whilst ATRA was previously found to broadly reduce Th17 responses, we found that antigen formulated in ATRA-containing cationic liposomes only inhibited Th17 cells in an antigen-specific manner and not when combined with an irrelevant antigen. Furthermore, this approach shifted existing Th17 cells away from IL-17A expression and transcriptomic analysis of sorted Th17 lineage cells from IL-17 fate reporter mice revealed a shift of antigen-specific Th17 cells to exTh17 cells, expressing functional markers associated with T cell regulation and tolerance. In the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, vaccination with myelin-specific (MOG) antigen in ATRA-containing liposomes reduced Th17 responses and alleviated disease. This highlights the potential of therapeutic vaccination for changing the phenotype of existing Th17 cells in the context of immune mediated diseases., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Successful oral delivery of liposomes requires formulations designed to withstand harsh gastrointestinal conditions, e.g., by converting to solid-state followed by loading into gastro-resistant delivery devices. The hypothesis was that the use of dextran-trehalose mixtures for spray drying would improve the rehydration kinetics of dried liposomes. The objectives were to determine the protective capacity of trehalose-dextran dehydration precursors and to increase the concentration of liposomes in the dry formulation volume. The study successfully demonstrated that 8.5% dextran combined with 76.5% trehalose protected CAF®04 liposomes during drying, with the liposome content maintained at 15% of the dry powder. Accordingly, the rehydration kinetics were slightly improved in formulations containing up to 8.5% dextran in the dry powder volume. Additionally, a 2.4-fold increase in lipid concentration (3 mM vs 7.245 mM) was achieved for spray dried CAF®04 liposomes. Ultimately, this study demonstrates the significance of trehalose as a primary carrier during spray drying of CAF®04 liposomes and highlights the advantage of incorporating small amounts of dextran to tune rehydration kinetics of spray-dried liposomes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: AL, GKP, STS are employed at Statens Serum Institut, which is a non-profit government research facility and holds patents on the CAF®-based adjuvants. All other authors declare that there are no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

CD4 T follicular helper cells (T fh ) are essential for establishing serological memory and have distinct helper attributes that impact both the quantity and quality of the antibody response. Insights into T fh subsets that promote antibody persistence and functional capacity can critically inform vaccine design. Based on the T fh profiles evoked by the live attenuated measles virus vaccine, renowned for its ability to establish durable humoral immunity, we investigated the potential of a T fh 1/17 recall response during the boost phase to enhance persistence of HIV-1 Envelope (Env) antibodies in rhesus macaques. Using a DNA-prime encoding gp160 antigen and T fh polarizing cytokines (interferon protein-10 (IP-10) and interleukin-6 (IL-6)), followed by a gp140 protein boost formulated in a cationic liposome-based adjuvant (CAF01), we successfully generated germinal center (GC) T fh 1/17 cells. In contrast, a similar DNA-prime (including IP-10) followed by gp140 formulated with monophosphoryl lipid A (MPLA) +QS-21 adjuvant predominantly induced GC T fh 1 cells. While the generation of GC T fh 1/17 cells with CAF01 and GC T fh 1 cells with MPLA +QS-21 induced comparable peak Env antibodies, the latter group demonstrated significantly greater antibody concentrations at week 8 after final immunization which persisted up to 30 weeks (gp140 IgG ng/ml- MPLA; 5500; CAF01, 2155; p<0.05). Notably, interferon γ +Env-specific T fh responses were consistently higher with gp140 in MPLA +QS-21 and positively correlated with Env antibody persistence. These findings suggest that vaccine platforms maximizing GC T fh 1 induction promote persistent Env antibodies, important for protective immunity against HIV., Competing Interests: AV, CH, SE, JS, DR, GP, XS, LW, GT, PK, RA, SI No competing interests declared, (© 2023, Verma, Hawes et al.)

Adjuvants are vaccine components that can boost the type, magnitude, breadth, and durability of an immune response. We have previously demonstrated that certain adjuvant combinations can act synergistically to enhance and shape immunogenicity including promotion of Th1 and cytotoxic T-cell development. These combinations also promoted protective immunity in vulnerable populations such as newborns. In this study, we employed combined antigen-specific human in vitro models to identify adjuvant combinations that could synergistically promote the expansion of vaccine-specific CD4 + cells, induce cross-presentation on MHC class I, resulting in antigen-specific activation of CD8 + cells, and direct the balance of immune response to favor the production of Th1-promoting cytokines. A screen of 78 adjuvant combinations identified several T cell-potentiating adjuvant combinations. Remarkably, a combination of TLR9 and STING agonists (CpG + 2,3-cGAMP) promoted influenza-specific CD4 + and CD8 + T cell activation and selectively favored production of Th1-polarizing cytokines TNF and IL-12p70 over co-regulated cytokines IL-6 and IL-12p40, respectively. Phenotypic reprogramming towards cDC1-type dendritic cells by CpG + 2,3-cGAMP was also observed. Finally, we characterized the molecular mechanism of this adjuvant combination including the ability of 2,3-cGAMP to enhance DC expression of TLR9 and the dependency of antigen-presenting cell activation on the Sec22b protein important to endoplasmic reticulum-Golgi vesicle trafficking. The identification of the adjuvant combination CpG + 2,3-cGAMP may therefore prove key to the future development of vaccines against respiratory viral infections tailored for the functionally distinct immune systems of vulnerable populations such as older adults and newborns., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: OL and SvH are named inventors on patents relating to vaccine adjuvants and in vitro systems that model human immunity. The rest of the authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Class-switching to IgG2a/c in mice is a hallmark response to intracellular pathogens. T cells can promote class-switching and the predominant pathway for induction of IgG2a/c antibody responses has been suggested to be via stimulation from Th1 cells. We previously formulated CAF®01 (cationic liposomes containing dimethyldioctadecylammonium bromide (DDA) and Trehalose-6,6-dibehenate (TDB)) with the lipidated TLR7/8 agonist 3M-052 (DDA/TDB/3M-052), which promoted robust Th1 immunity in newborn mice. When testing this adjuvant in adult mice using the recombinant Chlamydia trachomatis (C.t.) vaccine antigen CTH522, it similarly enhanced IgG2a/c responses compared to DDA/TDB, but surprisingly reduced the magnitude of the IFN-γ+Th1 response in a TLR7 agonist dose-dependent manner. Single-cell RNA-sequencing revealed that DDA/TDB/3M-052 liposomes initiated early transcription of class-switch regulating genes directly in pre-germinal center B cells. Mixed bone marrow chimeras further demonstrated that this adjuvant did not require Th1 cells for IgG2a/c switching, but rather facilitated TLR7-dependent T-bet programming directly in B cells. This study underlines that adjuvant-directed IgG2a/c class-switching in vivo can occur in the absence of T-cell help, via direct activation of TLR7 on B cells and positions DDA/TDB/3M-052 as a powerful adjuvant capable of eliciting type I-like immunity in B cells without strong induction of Th1 responses., (© 2023. The Author(s).)

Vaccines have relieved the public health burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and globally inactivated vaccines are most widely used. However, poor vaccination accessibility and waning immunity maintain the pandemic, driving emergence of variants. We developed an inactivated SARS-CoV-2 (I-SARS-CoV-2) vaccine based on a viral isolate with the Spike mutation D614G, produced in Vero cells in a scalable bioreactor, inactivated with β-propiolactone, purified by membrane-based steric exclusion chromatography, and adjuvanted with MF59-like adjuvant AddaVax. I-SARS-CoV-2 and a derived split vaccine induced persisting neutralizing antibodies in mice; moreover, lyophilized antigen was immunogenic. Following homologous challenge, I-SARS-CoV-2 immunized hamsters were protected against disease and lung pathology. In contrast with reports for widely used vaccines, hamster plasma similarly neutralized the homologous and the Delta (B.1.617.2) variant viruses, whereas the Omicron (B.1.1.529) variant was neutralized less efficiently. Applied bioprocessing approaches offer advantages regarding scalability and production, potentially benefitting worldwide vaccine coverage., Competing Interests: A.O., C.R.D.H., S.F., K.H., A.F.P., C.F-A., G.P.A., K.T.H., L.V.P., Y.Z., K.A.G., U.F., U.V.S., G.K.P., H.E.J., J.P.C., S.R., J.B., and J.M.G. declare no conflict of interests. P.M-G. is an inventor in pending patent applications related to the SXC purification method described in this work., (© 2023 The Authors.)

SARS-CoV-2 continues to pose a threat to human health as new variants emerge and thus a diverse vaccine pipeline is needed. We evaluated SARS-CoV-2 HexaPro spike protein formulated in Alhydrogel ® (aluminium oxyhydroxide) in Syrian hamsters, using an accelerated two dose regimen (given 10 days apart) and a standard regimen (two doses given 21 days apart). Both regimens elicited spike- and RBD-specific IgG antibody responses of similar magnitude, but in vitro virus neutralization was low or undetectable. Despite this, the accelerated two dose regimen offered reduction in viral load and protected against lung pathology upon challenge with homologous SARS-CoV-2 virus (Wuhan-Hu-1). This highlights that vaccine-induced protection against SARS-CoV-2 disease can be obtained despite low neutralizing antibody levels and suggests that accelerated vaccine schedules may be used to confer rapid protection against SARS-CoV-2 disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Christensen, Polacek, Sheward, Hanke, McInerney, Murrell, Hartmann, Jensen, Zimmermann, Jungersen, Illigen, Isling, Fernandez-Antunez, Ramirez, Bukh and Pedersen.)

Oral vaccination has in the recent years gained a lot of attraction, mainly due to optimized patient compliance and logistics. However, the development of oral vaccines, especially oral subunit vaccines is challenging. Micro technology can be utilized to overcome some of these challenges, by facilitating protection and effective delivery of the vaccine components in the gastrointestinal tract (GI tract). One such technology is Microcontainers (MCs), which can be realized to be mucoadhesive and to target specific regions of the GI tract via oral delivery. Here, we test MCs, for oral delivery of the C. trachomatis vaccine candidate CTH522, in combination with effective mucosal adjuvants. The adjuvants alpha- galactosylceramide (α-GalCer), C-di-GMP and cholera toxin B were compared in vivo, to identify the most prominent adjuvant for formulation with CTH522. Formulations were administered both purely oral and as boosters following a subcutaneous (s.c.) prime with CTH522 in combination with the CAF®01 adjuvant. CTH522 formulated with α-GalCer showed to be the most efficient combination for the oral vaccine, based on the immunological analysis. Lyophilized formulation of CTH522 and α-GalCer was loaded into MCs and these were subsequently coated with Eudragit L100-55 and evaluated in vivo in mice for the ability of MCs to mediate intestinal vaccine delivery and increase immunogenicity of the vaccine. Mice receiving oral prime and boosters did show a significantly enhanced mucosal immune responses compared to naive mice. This indicates the MCs are indeed capable of delivering the vaccine formulation intact and able to stimulate the immune cells. Mice orally boosted with MCs following a s.c. prime with CAF01, demonstrated improved systemic and local Th17 responses, along with increased local IFN-γ and IgA levels compared to both the s.c. prime alone and the homologous oral prime-boost immunization. However, due to the relatively weak observed effect of the MC delivery on the immune responses, it was hypothesized that the MCs are proportionally too large for the GI tract of mice, and thus cleared before an effective immune response can be induced. To investigate this, MCs were loaded with BaSO4, and orally administered to mice. Analysis with X-ray and CT showed a transit time of approximately 1-1.5 h from the stomach to the cecum, corresponding to the standard transit time in mice, and an extremely narrow absorption window. This indicates that mice is not a suitable animal model for evaluation of MCs. These data should be taken into consideration in future in vivo trials with this and similar technologies, where larger animals might be a necessity for proof-of-concept studies., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare, (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mice lacking the atypical inhibitory kappa B (IκB) protein, IκBNS, a regulator of the NF-κB pathway encoded by the nfkbid gene, display impaired antibody responses to both T cell-independent (TI) and T cell-dependent (TD) antigens. To better understand the basis of these defects, we crossed mice carrying floxed nfkbid alleles with mice expressing Cre under the transcriptional control of the Cd79a gene to create mice that lacked IκBNS expression only in B cells. Analyses of these conditional knock-out mice revealed intact CD4 + and CD8 + T cell populations, including preserved frequencies of FoxP3 + regulatory T cells, which are known to be reduced in IκBNS knock-out mice. Like IκBNS knock-out mice, mice with conditional IκBNS ablation in B cells displayed defective IgM responses to TI antigens and a severe reduction in peritoneal B-1a cells. However, in contrast to mice lacking IκBNS altogether, the conditional IκBNS knock-out mice responded well to TD antigens compared to the control mice, with potent IgG responses following immunization with the viral antigen, rSFV-βGal or the widely used hapten-protein model antigen, NP-CGG. Furthermore, B cell intrinsic IκBNS expression was dispensable for germinal center (GC) formation and T follicular helper cell responses to NP-CGG immunization. The results presented here suggest that the defect in antibody responses to TD antigens observed in IκBNS knock-out mice results from a B cell extrinsic defect., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Khoenkhoen, Ádori, Solís-Sayago, Soulier, Russell, Beutler, Pedersen and Karlsson Hedestam.)

Background: Licensed vaccines against SARS-CoV-2 effectively protect against severe disease, but display incomplete protection against virus transmission. Mucosal vaccines providing immune responses in the upper airways are one strategy to protect against transmission., Methods: We administered Spike HexaPro trimer formulated in a cationic liposomal adjuvant as a parenteral (subcutaneous - s.c.) prime - intranasal boost regimen to elicit airway mucosal immune responses and evaluated this in a Syrian hamster model of virus transmission., Findings: Parenteral prime - intranasal boost elicited high-magnitude serum neutralizing antibody responses and IgA responses in the upper respiratory tract. The vaccine strategy protected against virus in the lower airways and lung pathology, but virus could still be detected in the upper airways. Despite this, the parenteral prime - intranasal booster vaccine effectively protected against onward SARS-CoV-2 transmission., Interpretation: This study suggests that parenteral-prime mucosal boost is an effective strategy for protecting against SARS-CoV-2 infection and highlights that protection against virus transmission may be obtained despite incomplete clearance of virus from the upper respiratory tract. It should be noted that protection against onward transmission was not compared to standard parenteral prime-boost, which should be a focus for future studies., Funding: This work was primarily supported by the European Union Horizon 2020 research and innovation program under grant agreement no. 101003653., Competing Interests: Declaration of interests D.C. is co-inventor on patents on the cationic adjuvant formulations (CAF). All rights have been turned over to Statens Serum Institut, which is a non-profit government research facility. The rest of the authors declare that there are no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Respiratory syncytial virus is a leading cause of morbidity and mortality in children, due in part to their distinct immune system, characterized by impaired induction of Th 1 immunity. Here we show application of cationic adjuvant formulation CAF08, a liposomal vaccine formulation tailored to induce Th 1 immunity in early life via synergistic engagement of Toll-like Receptor 7/8 and the C-type lectin receptor Mincle. We apply quantitative phosphoproteomics to human dendritic cells and reveal a role for Protein Kinase C-δ for enhanced Th1 cytokine production in neonatal dendritic cells and identify signaling events resulting in antigen cross-presentation. In a murine in vivo model a single immunization at birth with CAF08-adjuvanted RSV pre-fusion antigen protects newborn mice from RSV infection by induction of antigen-specific CD8 + T-cells and Th1 cells. Overall, we describe a pediatric adjuvant formulation and characterize its mechanism of action providing a promising avenue for development of early life vaccines against RSV and other respiratory viral pathogens., (© 2022. The Author(s).)

Chlamydia trachomatis is the most prevalent sexually transmitted disease of bacterial origin. The high number of asymptomatic cases makes it difficult to stop the transmission, requiring vaccine development. Herein, a strategy is proposed to obtain local genital tract immunity against C. trachomatis through parenteral prime and sublingual boost. Subcutaneous administration of chlamydia CTH522 subunit vaccine loaded in the adjuvant CAF01 is combined with sublingual administration of CTH522 loaded in a novel thermosensitive and mucoadhesive hydrogel. Briefly, a ternary optimized hydrogel (OGEL) with desirable biological and physicochemical properties is obtained using artificial intelligence techniques. This formulation exhibits a high gel strength and a strong mucoadhesive, adhesive and cohesive nature. The thermosensitive properties of the hydrogel facilitate application under the tongue. Meanwhile the fast gelation at body temperature together with rapid antigen release should avoid CTH522 leakage by swallowing and increase the contact with sublingual tissue, thus promoting absorption. In vivo studies demonstrate that parenteral-sublingual prime-boost immunization, using CAF01 and OGEL as CTH522 vaccine carriers, shows a tendency to increase cellular (Th1/Th17) immune responses when compared to mucosal or parenteral vaccination alone. Furthermore, parenteral prime with CAF01/CTH522 followed by sublingual boosting with OGEL/CTH522 elicits a local IgA response in the genital tract., (© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

The SARS-CoV-2 pandemic caused a massive health and societal crisis, although the fast development of effective vaccines reduced some of the impact. To prepare for future respiratory virus pandemics, a pan-viral prophylaxis could be used to control the initial virus outbreak in the period prior to vaccine approval. The liposomal vaccine adjuvant CAF ® 09b contains the TLR3 agonist polyinosinic:polycytidylic acid, which induces a type I interferon (IFN-I) response and an antiviral state in the affected tissues. When testing CAF09b liposomes as a potential pan-viral prophylaxis, we observed that intranasal administration of CAF09b liposomes to mice resulted in an influx of innate immune cells into the nose and lungs and upregulation of IFN-I-related gene expression. When CAF09b liposomes were administered prior to challenge with mouse-adapted influenza A/Puerto Rico/8/1934 virus, it protected from severe disease, although the virus was still detectable in the lungs. However, when CAF09b liposomes were administered after influenza challenge, the mice had a similar disease course to controls. In conclusion, CAF09b may be a suitable candidate as a pan-viral prophylactic treatment for epidemic viruses, but must be administered prior to virus exposure to be effective., Competing Interests: All authors except R.J.C. and F.Z. are employed by Statens Serum Institut, a nonprofit government research facility, which holds patents on the cationic liposomal adjuvants (CAF). F.F. and D.C. are co-inventors on a patent application related to the technology application.

Antibodies binding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike have therapeutic promise, but emerging variants show the potential for virus escape. This emphasizes the need for therapeutic molecules with distinct and novel neutralization mechanisms. Here we describe the isolation of a nanobody that interacts simultaneously with two RBDs from different spike trimers of SARS-CoV-2, rapidly inducing the formation of spike trimer-dimers leading to the loss of their ability to attach to the host cell receptor, ACE2. We show that this nanobody potently neutralizes SARS-CoV-2, including the beta and delta variants, and cross-neutralizes SARS-CoV. Furthermore, we demonstrate the therapeutic potential of the nanobody against SARS-CoV-2 and the beta variant in a human ACE2 transgenic mouse model. This naturally elicited bispecific monomeric nanobody establishes an uncommon strategy for potent inactivation of viral antigens and represents a promising antiviral against emerging SARS-CoV-2 variants., (© 2022. The Author(s).)

The Paleocene-Eocene Thermal Maximum (PETM) was a period of geologically-rapid carbon release and global warming ~56 million years ago. Although modelling, outcrop and proxy records suggest volcanic carbon release occurred, it has not yet been possible to identify the PETM trigger, or if multiple reservoirs of carbon were involved. Here we report elevated levels of mercury relative to organic carbon-a proxy for volcanism-directly preceding and within the early PETM from two North Sea sedimentary cores, signifying pulsed volcanism from the North Atlantic Igneous Province likely provided the trigger and subsequently sustained elevated CO 2 . However, the PETM onset coincides with a mercury low, suggesting at least one other carbon reservoir released significant greenhouse gases in response to initial warming. Our results support the existence of 'tipping points' in the Earth system, which can trigger release of additional carbon reservoirs and drive Earth's climate into a hotter state., (© 2021. The Author(s).)

The degree of antigen adsorption to adjuvants in subunit vaccines may significantly influence the immune responses they induce upon vaccination. Commonly used approaches for studying how the level of adsorption affects the induction of antigen-specific immune responses include (i) using adjuvants with different abilities to adsorb antigens, and (ii) comparing different antigens selected based on their ability to adsorb to the adjuvant. A weakness of these approaches is that not only the antigen adsorption level is varied, but also other important functional factors such as adjuvant composition and/or the B/T cell epitopes, which may affect immunogenicity. Hence, we investigated how changing the adsorption capabilities of a single antigen to an adjuvant influenced the vaccine-induced immune responses. The model antigen lysozyme, which displays a positive net charge at physiological pH due to an isoelectric point (pI) of 11, was succinylated to different extents, resulting in a reduction of the pI value to 4.4-5.9, depending on the degree of succinylation. A pronounced inverse correlation was found between the pI value of the succinylated lysozyme analogues and the degree of adsorption to a cationic liposomal adjuvant consisting of dimethyldioctadecylammonium bromide (DDA) and trehalose dibehenate (TDB) (CAF®01). Furthermore, increased adsorption to this adjuvant correlated directly with the magnitude of lysozyme-specific Th1/Th17 immune responses induced by the vaccine in mice, while there was an inverse correlation with antibody induction. However, high lysozyme-specific antibody titers were induced with an increased antigen dose, even upon vaccination with a strongly adsorbed succinylated lysozyme analogue. Hence, these data illustrate that the degree of lysozyme adsorption to CAF®01 strongly affects the quality of the resulting immune responses., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

One of the main goals of vaccine research is the development of adjuvants that can enhance immune responses and are both safe and biocompatible. We explored the application of the natural polymer hyaluronan (HA) as a promising immunological adjuvant for protein-based vaccines. Chemical conjugation of HA to antigens strongly increased their immunogenicity, reduced booster requirements, and allowed antigen dose sparing. HA-based bioconjugates stimulated robust and long-lasting humoral responses without the addition of other immunostimulatory compounds and proved highly efficient when compared to other adjuvants. Due to its intrinsic biocompatibility, HA allowed the exploitation of different injection routes and did not induce inflammation at the inoculation site. This polymer promoted rapid translocation of the antigen to draining lymph nodes, thus facilitating encounters with antigen-presenting cells. Overall, HA can be regarded as an effective and biocompatible adjuvant to be exploited for the design of a wide variety of vaccines.

Background: SARS-CoV-2 has caused a global pandemic, infecting millions of people. A safe, effective vaccine is urgently needed and remains a global health priority. Subunit vaccines are used successfully against other viruses when administered in the presence of an effective adjuvant., Methods: We evaluated three different clinically tested adjuvant systems in combination with the SARS-CoV-2 pre-fusion stabilized (S-2P) spike protein using a one-dose regimen in mice., Findings: Whilst spike protein alone was only weakly immunogenic, the addition of either Aluminum hydroxide, a squalene based oil-in-water emulsion system (SE) or a cationic liposome-based adjuvant significantly enhanced antibody responses against the spike receptor binding domain (RBD). Kinetics of antibody responses differed, with SE providing the most rapid response. Neutralizing antibodies developed after a single immunization in all adjuvanted groups with ID 50 titers ranging from 86-4063. Spike-specific CD4 T helper responses were also elicited, comprising mainly of IFN-γ and IL-17 producing cells in the cationic liposome adjuvanted group, and more IL-5- and IL-10-secreting cells in the AH group., Interpretation: These results demonstrate that adjuvanted spike protein subunit vaccine is a viable strategy for rapidly eliciting SARS-CoV-2 neutralizing antibodies and CD4 T cell responses of various qualities depending on the adjuvant used, which can be explored in further vaccine development against COVID-19., Funding: This work was supported by the European Union Horizon 2020 research and innovation program under grant agreement no. 101003653., Competing Interests: Declaration of Competing Interest D.C. is co-inventor on patents on the cationic adjuvant formulations (CAF). All rights have been turned over to Statens Serum Institut, which is a non-profit government research facility. The rest of the authors declare that there are no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Humoral immunity is established after differentiation of antigen-specific B cells into plasma cells (PCs) that produce antibodies of relevant specificities. Defects in the development, activation, or differentiation of B cells severely compromises the immune response. Primary immunodeficiencies are often characterized by hypogammaglobulinemia and the inability to mount effective antigen-specific antibody responses, resulting in increased susceptibility to infections. After IgA deficiency, which is most often asymptomatic, common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency, but in most cases the underlying genetic causes are unknown or their roles in disease pathogenesis are poorly understood. In this study, we developed a protocol for in vitro stimulation of primary human B cells for subsequent analyses of PC differentiation and antibody production. With this approach, we were able to detect a population of CD38 + IRF4 + Blimp-1 + cells committed to PC fate and IgG production, including when starting from cryopreserved samples. The application of functional assays to characterize PC differentiation and possible defects therein in B cells from patients suffering from primary antibody deficiencies with late B cell defects could increase our understanding of the disease pathophysiology and underlying mechanisms., (Copyright © 2020 Khoenkhoen, Ádori, Pedersen and Karlsson Hedestam.)

Aluminum salts and squalene based oil-in-water emulsions (SE) are widely used adjuvants in licensed vaccines, yet their mechanisms are not fully known. Here we report that induction of antibody responses displays different kinetics dependent on the adjuvant used. SE facilitated a rapid antibody response in contrast to aluminum hydroxide (AH) and the depot-forming cationic liposome-based adjuvant (CAF01). Antigen given with the SE adjuvant rapidly reached follicular B cells in the draining lymph node, whereas antigen formulated in AH or CAF01 remained at the site of injection as a depot. Removal of the injection site early after immunization abrogated antibody responses only when antigen was given in the depot-forming adjuvants. Despite initial delays in B cell activation and germinal center (GC) formation when antigen was given in depot-forming adjuvants, the antibody levels reached higher magnitudes than when the antigen was formulated in SE. This study demonstrates that the kinetic aspect of antibody responses is critical in adjuvant benchmarking and suggests that the optimal vaccination regime depends on the adjuvant used., (Copyright © 2020 Pedersen, Wørzner, Andersen and Christensen.)

Therapeutic vaccination offers great promise as an intervention for a diversity of infectious and non-infectious conditions. Given that most chronic health conditions are thought to have an immune component, vaccination can at least in principle be proposed as a therapeutic strategy. Understanding the nature of protective immunity is of vital importance, and the progress made in recent years in defining the nature of pathological and protective immunity for a range of diseases has provided an impetus to devise strategies to promote such responses in a targeted manner. However, in many cases, limited progress has been made in clinical adoption of such approaches. This in part results from a lack of safe and effective vaccine adjuvants that can be used to promote protective immunity and/or reduce deleterious immune responses. Although somewhat simplistic, it is possible to divide therapeutic vaccine approaches into those targeting conditions where antibody responses can mediate protection and those where the principal focus is the promotion of effector and memory cellular immunity or the reduction of damaging cellular immune responses as in the case of autoimmune diseases. Clearly, in all cases of antigen-specific immunotherapy, the identification of protective antigens is a vital first step. There are many challenges to developing therapeutic vaccines beyond those associated with prophylactic diseases including the ongoing immune responses in patients, patient heterogeneity, and diversity in the type and stage of disease. If reproducible biomarkers can be defined, these could allow earlier diagnosis and intervention and likely increase therapeutic vaccine efficacy. Current immunomodulatory approaches related to adoptive cell transfers or passive antibody therapy are showing great promise, but these are outside the scope of this review which will focus on the potential for adjuvanted therapeutic active vaccination strategies., (© 2020 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.)

Conserved epitopes are targets commonly researched to be part of universal vaccine candidates against influenza viruses (IV). These conserved epitopes need to be cross-protecting against distinct IV subtypes and to have a strong immunogenic potential. Nevertheless, subunit vaccines generally require a strong adjuvant to enhance their immunological effects. Herewith, we compare four different adjuvants differing in their immunological signatures that may enhance efficacy of a conserved hemagglutinin (HA)-epitope from IV, the NG-34, to define the most efficient combination of antigen/adjuvant to combat IV infections. Soluble NG-34 was mixed with adjuvants like aluminium hydroxide (AH) and AddaVax, known to induce Th2 and humoral responses; CAF01 which displays a biased Th1/Th17 profile and Diluvac Forte which augments the humoral response. Combinations were tested in different groups of mice which were subjected to immunological analyses. CAF01 + NG-34 induced a complete immune response with the highest IgG1, IgG2c titers and percentages of activated CD4 T cell promoting IFN-γ, IL-2 and TNF-α producing cells. Furthermore, in NG-34 stimulated mice splenocytes, cytokine levels of IFN-γ, IL-1β, IL-6, IL-10, IL-17 and TNF-α were also the highest in the CAF01 + NG-34 mouse group. This complete induced immune response covering the humoral and the cellular arms of the adaptive immunity promoted by CAF01 + NG-34 group suggests that CAF01 could be a good candidate as an adjuvant to combine with NG-34 for an efficacious vaccine against IV. However, more studies performed in IV hosts as well as studies with a challenge model are further required.

A major roadblock in the development of novel vaccines is the formulation and delivery of the antigen. Liposomes composed of a dimethyldioctadecylammonium (DDA) backbone and the adjuvant trehalose-6-6-dibehenate (TDB, termed "cationic adjuvant formulation (CAF01)", promote immunogenicity and protective efficacy of vaccines, most notably against infection with Mycobacterium tuberculosis. Specifically, the multicomponent antigen H56 delivered by CAF01 protects against tuberculosis in mice. Here we investigated whether the inclusion of immune-modulatory adjuvants into CAF01 modulates the immunogenicity of H56/CAF01 in vitro and in vivo. Based on our recent findings we selected the active sequence of the mycobacterial 19 kDa lipoprotein, Pam 3 Cys, which interacts with Toll like receptor 2 to induce an antimicrobial pathway. H56/CAF01-Pam 3 Cys liposomes were characterized for Pam 3 Cys incorporation, size, toxicity and activation of primary human macrophages. Macrophages efficiently take up H56/CAF01-Pam 3 Cys and trigger the release of significantly higher levels of TNF, IL-12 and IL-10 than H56/CAF01 alone. To evaluate the immunogenicity in vivo, we immunized mice with H56/CAF01-Pam 3 Cys and measured the release of IFN-γ and IL-17A by lymph node cells and spleen cells. While the antigen-specific production of IFN-γ was reduced by inclusion of Pam 3 Cys into H56/CAF01, the levels of IL-17A remained unchanged. In agreement with this finding, the concentration of the IFN-γ-associated IgG2a antibodies in the serum was lower than in H56/CAF01 immunized animals. These results provide proof of concept that Toll like-receptor agonist can be included into liposomes to modulate immune responses. The discordant results between the in vitro studies with human macrophages and in vivo studies in mice highlight the relevance and complexity of comparing immune responses in different species.

During the past 600 million years of Earth history, four of five major extinction events were synchronous with volcanism in large igneous provinces. Despite improved temporal frameworks for these events, the mechanisms causing extinctions remain unclear. Volcanic emissions of greenhouse gases, SO 2 , and halocarbons are generally considered as major factors in the biotic crises, resulting in global warming, acid deposition, and ozone layer depletion. Here, we show that pulsed elevated concentrations of mercury in marine and terrestrial sediments across the Triassic-Jurassic boundary in southern Scandinavia and northern Germany correlate with intense volcanic activity in the Central Atlantic Magmatic Province. The increased levels of mercury-the most genotoxic element on Earth-also correlate with high occurrences of abnormal fern spores, indicating severe environmental stress and genetic disturbance in the parent plants. We conclude that this offers compelling evidence that emissions of toxic volcanogenic substances contributed to the end-Triassic biotic crisis., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Impaired classical NF-κB pathway signaling causes reduced antibody responses to T-independent (TI) antigens. We investigated the potential reasons for defective TI responses in mice lacking the atypical inhibitory kappa B (IκB) protein of the NF-κB pathway, IκBNS. Analyses of the plasma cell compartment in vitro and in vivo after challenge with lipopolysaccharide (LPS) showed significant decreases in the frequencies of plasma cells in the absence of IκBNS. In vitro activation of B cells via the B cell receptor or via Toll-like receptor 4 revealed that early activation events were unaffected in IκBNS-deficient B cells, while proliferation was reduced compared to in similarly stimulated wildtype (wt) B cells. IκBNS-deficient B cells also displayed impaired upregulation of the transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), which is essential for TI responses, and decreased sensitivity to TACI ligands upon stimulation. Furthermore, IκBNS-deficient B cells, in contrast to wt B cells, displayed altered expression of IRF4, Blimp-1 and Pax5 upon LPS-induced differentiation, indicating impaired transcriptional regulation of plasma cell generation., (© 2018 The Authors Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australasian Society for Immunology Inc.)

Many different adjuvants are currently being developed for subunit vaccines against a number of pathogens and diseases. Rational design is increasingly used to develop novel vaccine adjuvants, which requires extensive knowledge of, for example, the desired immune responses, target antigen-presenting cell subsets, their localization, and expression of relevant pattern-recognition receptors. The adjuvant mechanism of action and efficacy are usually evaluated in animal models, where mice are by far the most used. In this review, we present methods for assessing adjuvant efficacy and function in animal models: (1) whole-body biodistribution evaluated by using fluorescently and radioactively labeled vaccine components; (2) association and activation of immune cell subsets at the injection site, in the draining lymph node, and the spleen; (4) adaptive immune responses, such as cytotoxic T-lymphocytes, various T-helper cell subsets, and antibody responses, which may be quantitatively evaluated using ELISA, ELISPOT, and immunoplex assays and qualitatively evaluated using flow cytometric and single cell sequencing assays; and (5) effector responses, for example, antigen-specific cytotoxic potential of CD8+ T cells and antibody neutralization assays. While the vaccine-induced immune responses in mice often correlate with the responses induced in humans, there are instances where immune responses detected in mice are not translated to the human situation. We discuss some examples of correlation and discrepancy between mouse and human immune responses and how to understand them., (© The Author(s) 2019. Published by Oxford University Press on behalf of the National Academy of Sciences. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Pattern recognition receptors, including the Toll-like receptors (TLRs), are important in the induction and activation of two critical arms of the host defence to pathogens and microorganisms: the rapid innate immune response (as characterised by the production of Th1 promoting cytokines and type 1 interferons) and the adaptive immune response. Through this activation, ligands and agonists of TLRs can enhance immunotherapeutic efficacy. Resiquimod is a small (water-soluble) agonist of the endosome-located Toll-like receptors 7 and 8 (TLR7/8). However due to its molecular attributes it rapidly distributes throughout the body after injection. To circumvent this, these TLR agonists can be incorporated within delivery systems, such as liposomes, to promote the co-delivery of both antigen and agonists to antigen presenting cells. In this present study, resiquimod has been chemically conjugated to a lipid to form a lipid-TLR7/8 agonist conjugate which can be incorporated within immunogenic cationic liposomes composed of dimethyldioctadecylammonium bromide (DDA) and the immunostimulatory glycolipid trehalose 6,6' - dibehenate (TDB). This DDA:TDB-TLR7/8 formulation offers similar vesicle characteristics to DDA:TDB (size and charge) and offers high retention of both resiquimod and the electrostatically adsorbed TB subunit antigen Ag85B-ESAT6-Rv2660c (H56). Following immunisation through the intramuscular (i.m.) route, these cationic DDA:TDB-TLR7/8 liposomes form a vaccine depot at the injection site. However, immunisation studies have shown that this biodistribution does not translate into notably increased antibody nor Th1 responses at the spleen and draining popliteal lymph node compared to DDA:TDB liposomes. This work demonstrates that the conjugation of TLR7/8 agonists to cationic liposomes can promote co-delivery but the immune responses stimulated do not merit the added complexity considerations of the formulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The development of the CAF family adjuvant was initiated around 20 years ago when Statens Serum Institut was preparing its first generation protein based recombinant subunit vaccine against tuberculosis for clinical testing, but realized that there were no clinically relevant adjuvants available that would support the strong CMI response needed. Since then the aim for the adjuvant research at Statens Serum Institut has been to provide adjuvants with distinct immunogenicity profiles correlating with protection for any given infectious disease. Two of the adjuvants CAF01 and CAF09 are currently being evaluated in human clinical trials. The purpose of this review is to give an overview of the immunocorrelates of those CAF adjuvants furthest in development. We further aim at giving an overview of the mechanism of action of the CAF adjuvants., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

B-1a cells are mainly generated from fetal liver progenitor cells, peri- and neonatally. The developmental steps and anatomical sites required for these cells to become mature B-1a cells remain elusive. We recently described a phenotypically distinct transitional B cell subset in the spleen of neonatal mice that generated B-1a cells when adoptively transferred. This, in combination with findings demonstrating that B-1a cells are lacking in congenitally asplenic mice, led us to hypothesize that the neonatal spleen is required for B-1a cell development. In accordance with previous reports, we found that B-1a cell numbers were reduced in adult mice that had undergone splenectomy compared to after sham surgery. In contrast, neonatal splenectomy led to peritoneal B-1a cell frequencies comparable to those observed in sham-operated mice until 6 weeks after surgery, suggesting that an intact spleen is required for B-1a cell maintenance rather than development. To study the role of the prenatal spleen in generating B-1a cells, we transferred fetal liver cells from pre-splenic embryos [embryonic age 11 (E11) days] into splenectomized recipient mice. B-1a cells were generated in the absence of the spleen, albeit at slightly reduced frequencies, and populated the peritoneal cavity and bone marrow. Lower bone marrow B-1a cell frequencies were also observed both after neonatal and adult splenectomy. These results demonstrated that B-1a cells could be generated in the complete absence of an intact spleen, but that asplenia led to a decline in these cells, suggesting a role of the spleen for maintaining the B-1a compartment.

There is an unmet medical need for new subunit vaccines that induce cytotoxic T-lymphocyte (CTL) responses to prevent infection with a number of pathogens. However, stimulation of CTL responses via clinically acceptable subcutaneous (s.c.) and intramuscular (i.m.) injection is challenging. Recently, we designed a liposomal adjuvant [cationic adjuvant formulation (CAF)09] composed of the cationic lipid dimethyldioctadecylammonium (DDA) bromide, a synthetic monomycoloyl glycerol analog and polyinosinic:polycytidylic acid, which induce strong CTL responses to peptide and protein antigens after intraperitoneal administration. By contrast, CAF09 does not stimulate CTL responses upon s.c. or i.m. injection because the vaccine forms a depot that remains at the injection site. Hence, we engineered a series of nanoemulsions (CAF24a-c) based on the active components of CAF09. The oil phase consisted of biodegradable squalane, and the surface charge was varied systematically by replacing DDA with zwitterionic distearoylphosphoethanolamine. We hypothesized that the nanoemulsions drain to the lymph nodes to a larger extent than CAF09, upon s.c. co-administration with the model antigen chicken egg ovalbumin (OVA). This results in an increased dose fraction that reaches the draining lymph nodes (dLNs) and subsequently activates cross-presenting dendritic cells (DCs), which can prime CTL responses. Indeed, the nanoemulsions induced antigen-specific CD8 + T-cell responses, which were significantly higher than those stimulated by OVA adjuvanted with CAF09. We explain this by the observed rapid localization of CAF24a in the dLNs and the subsequent association with conventional DCs, which promotes induction of CTL responses. Uptake of CAF24a was not specific for DCs, because CAF24a was also detected with B cells and macrophages. No measurable dose fraction of CAF09 was detected in the dLNs within the study period, and CAF09 formed a depot at the site of injection. Importantly, s.c. vaccination with OVA adjuvanted with CAF24a induced significant levels of specific lysis of antigen-pulsed splenocytes were induced after, which was not observed for OVA adjuvanted with CAF09. Thus, CAF24a is a promising adjuvant for induction of CTL responses upon s.c. and i.m. immunization, and it offers interesting perspectives for the design of vaccines against pathogens for which CTL responses are required to prevent infection.

Marginal zone (MZ) B cells reside in the splenic MZ and play important roles in T cell-independent humoral immune responses against blood-borne pathogens. IκBNS-deficient bumble mice exhibit a severe reduction in the MZ B compartment but regain an MZ B population with age and, thus, represent a valuable model to examine the biology of MZ B cells. In this article, we characterized the MZ B cell defect in further detail and investigated the nature of the B cells that appear in the MZ of aged bumble mice. Flow cytometry analysis of the splenic transitional B cell subsets demonstrated that MZ B cell development was blocked at the transitional-1 to transitional-2-MZ precursor stage in the absence of functional IκBNS. Immunohistochemical analysis of spleen sections from wild-type and bumble mice revealed no alteration in the cellular MZ microenvironment, and analysis of bone marrow chimeras indicated that the MZ B cell development defect in bumble mice was B cell intrinsic. Further, we demonstrate that the B cells that repopulate the MZ in aged bumble mice were distinct from age-matched wild-type MZ B cells. Specifically, the expression of surface markers characteristic for MZ B cells was altered and the L chain Igλ + repertoire was reduced in bumble mice. Finally, plasma cell differentiation of sorted LPS-stimulated MZ B cells was impaired, and aged bumble mice were unable to respond to NP-Ficoll immunization. These results demonstrate that IκBNS is required for an intact MZ B cell compartment in C57BL/6 mice., (Copyright © 2018 by The American Association of Immunologists, Inc.)

B cell receptor signaling and downstream NF-κB activity are crucial for the maturation and functionality of all major B cell subsets, yet the molecular players in these signaling events are not fully understood. Here we use several genetically modified mouse models to demonstrate that expression of the multifunctional BRCT (BRCA1 C-terminal) domain-containing PTIP (Pax transactivation domain-interacting protein) chromatin regulator is controlled by B cell activation and potentiates steady-state and postimmune antibody production in vivo. By examining the effects of PTIP deficiency in mice at various ages during ontogeny, we demonstrate that PTIP promotes bone marrow B cell development as well as the neonatal establishment and subsequent long-term maintenance of self-reactive B-1 B cells. Furthermore, we find that PTIP is required for B cell receptor- and T:B interaction-induced proliferation, differentiation of follicular B cells during germinal center formation, and normal signaling through the classical NF-κB pathway. Together with the previously identified role for PTIP in promoting sterile transcription at the Igh locus, the present results establish PTIP as a licensing factor for humoral immunity that acts at several junctures of B lineage maturation and effector cell differentiation by controlling B cell activation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

The need for more effective influenza vaccines is highlighted by the emergence of novel influenza strains, which can lead to new pandemics. There is a growing population of susceptible subjects at risk for severe complications of influenza, such as the elderly who are only in part protected by current licensed seasonal vaccines. One strategy for improving seasonal and pandemic vaccines takes advantage of adjuvants to boost and modulate evoked immune responses. In this study, we examined the capacity of the recently described adjuvant cyclic di-adenosine monophosphate (c-di-AMP) to serve as an adjuvant for improved mucosal influenza vaccines, and induce effective protection against influenza H5N1. In detail, c-di-AMP promoted (i) effective local and systemic humoral immune responses, including protective hemagglutination inhibition titers, (ii) effective cellular responses, including multifunctional T cell activity, (iii) induction of long-lasting immunity, and (iv) protection against viral challenge. Furthermore, we demonstrated the dose-sparing capacity of the adjuvant as well as the ability to evoke cross-clade protective immune responses. Overall, our results suggest that c-di-AMP contributes to the generation of a protective cell-mediated immune response required for efficacious vaccination against influenza, which supports the further development of c-di-AMP as an adjuvant for seasonal and pandemic influenza mucosal vaccines.

Mice deficient in central components of classical NF-κB signaling have low levels of circulating natural IgM antibodies and fail to respond to immunization with T-independent type 2 (TI-2) antigens. A plausible explanation for these defects is the severely reduced numbers of B-1 and marginal zone B (MZB) cells in such mice. By using an ethyl-N-nitrosourea mutagenesis screen, we identified a role for the atypical IκB protein IκBNS in humoral immunity. IκBNS-deficient mice lack B-1 cells and have severely reduced numbers of MZB cells, and thus resemble several other strains with defects in classical NF-κB signaling. We analyzed mice heterozygous for the identified IκBNS mutation and demonstrate that these mice have an intermediary phenotype in terms of levels of circulating IgM antibodies and responses to TI-2 antigens. However, in contrast to mice that are homozygous for the IκBNS mutation, the heterozygous mice had normal frequencies of B-1 and MZB cells. These results suggest that there is a requirement for IκBNS expression from two functional alleles for maintaining normal levels of circulating natural IgM antibodies and responses to TI-2 antigens.

NF-κB transcription factors play essential roles in hematopoiesis. In this review, we summarize the requirements of different components of the NF-κB pathway for B-1 cell development and maintenance. The B-1 cell developmental steps are also reviewed, with particular emphasis on stages where NF-κB signaling may be critical., (© 2015 New York Academy of Sciences.)

Signalling through Toll-like receptors (TLRs) by endogenous components of viruses or bacteria can promote antibody (Ab) isotype switching to IgG2a/c. Multiple cell types are capable of responding to TLR stimulation in vivo and the processes underlying TLR-induced Ab isotype switching are not fully defined. Here, we used feeble mice, which are deficient in the peptide/histidine transporter solute carrier family 15 member 4 (Slc15a4), and fail to produce cytokines including interferon alpha (IFNα) in response to TLR9 stimulation, to study Ab isotype switching to IgG2c in response to vaccination. We demonstrate that the production of IgG2c in response to CpGA-adjuvanted vaccines was severely reduced in feeble mice, while a more subtle defect was observed for CpGB. The reduced IgG2c production in feeble could not be ascribed to defective plasmacytoid dendritic cell (pDC) responses alone as we found that splenic cDCs and B cells from feeble mice were also defective in response to TLR9 ligation ex vivo. We conclude that Slc15a4 is required for intact function of TLR9-expressing cells and for effective Ab isotype switching to IgG2c in response to CpG-adjuvanted vaccines.

In this paper we document Fagaceae pollen from the Eocene of western Greenland. The pollen record suggests a remarkable diversity of the family in the early Cenozoic of Greenland. Extinct Fagaceae pollen types include Eotrigonobalanus , which extends at least back to the Paleocene, and two ancestral pollen types with affinities to the Eurasian Quercus Group Ilex and the western North American Quercus Group Protobalanus. In addition, modern lineages of Fagaceae are unambiguously represented by pollen of Fagus, Quercus Group Lobatae/Quercus, and three Castaneoideae pollen types. These findings corroborate earlier findings from Axel Heiberg Island that Fagaceae were a dominant element at high latitudes during the early Cenozoic. Comparison with coeval or older mid-latitude records of modern lineages of Fagaceae shows that modern lineages found in western Greenland and Axel Heiberg likely originated at lower latitudes. Further examples comprise (possibly) Acer , Aesculus , Alnus , Ulmus , and others. Thus, before fossils belonging to modern northern temperate lineages will have been recovered from older (early Eocene, Paleocene) strata from high latitudes, Engler's hypothesis of an Arctic origin of the modern temperate woody flora of Eurasia, termed 'Arcto-Tertiary Element', and later modification by R. W. Chaney and H. D. Mai ('Arcto-Tertiary Geoflora') needs to be modified.

B-1 cells mediate early protection against infection by responding to T cell-independent (TI) antigens found on the surface of various pathogens. Mice with impaired expression of the atypical IκB protein IκBNS have markedly reduced frequencies of B-1 cells. We used a mouse strain with dysfunctional IκBNS derived from an N-ethyl-N-nitrosourea (ENU) screen, named bumble, to investigate the point in the development of B-1 cells where IκBNS is required. The presence of wild-type (wt) peritoneal cells in mixed wt/bumble chimeras did not rescue the development of bumble B-1 cells, but wt peritoneal cells transferred to bumble mice restored natural IgM levels and response to TI antigens. The bumble and wt mice displayed similar levels of fetal liver B-1 progenitors and splenic neonatal transitional B (TrB) cells, both of which were previously shown to give rise to B-1 cells. Interestingly, we found that a subset of wt neonatal TrB cells expressed common B-1a markers (TrB-1a) and that this cell population was absent in the bumble neonatal spleen. Sorted TrB-1a (CD93(+)IgM(+)CD5(+)) cells exclusively generated B-1a cells when adoptively transferred, whereas sorted CD93(+)IgM(+)CD5(-) cells gave rise to B-2 cells and, to a lesser extent, B-1b and B-1a cells. This study identifies a phenotypically distinct splenic population of TrB-1a cells and establishes that the development of B-1a cells is blocked before this stage in the absence of IκBNS.

Continued efforts to define the immunogenic properties of the HIV-1 envelope glycoproteins (Env) are needed to elicit effective antibody (Ab) responses by vaccination. HIV-1 is a highly neutralization-resistant virus due to conformational and glycan shielding of conserved Ab determinants on the virus spike. Elicitation of broadly neutralizing Abs that bind poorly accessible epitope regions on Env is therefore extremely challenging and will likely require selective targeting of specific sub-determinants. To evaluate such approaches there is a pressing need for in vivo studies in both large and small animals, including mice. Currently, most mouse immunization studies are performed in the BALB/c strain; however, the C57BL/6 strain offers improved possibilities for mechanistic studies due to the availability of numerous knock-out strains on this genetic background. Here, we compared Env immunogenicity in BALB/c and C57BL/6 mice and found that the magnitude of the antigen-specific response was somewhat lower in C57BL/6 than in BALB/c mice by ELISA but not significantly different by B cell ELISpot measurements. We then established protocols for the isolation of single Env-specific memory B cells and germinal center (GC) B cells from immunized C57BL/6 mice to facilitate future studies of the elicited response at the monoclonal Ab level. We propose that these protocols can be used to gain an improved understanding of the early recruitment of Env-specific B cells to the GC as well as the archiving of such responses in the memory B cell pool following immunization.

Background: Influenza H5N1 virus constitutes a pandemic threat and development of effective H5N1 vaccines is a global priority. Anti-influenza antibodies directed towards the haemagglutinin (HA) define a correlate of protection. Both antibody concentration and avidity may be important for virus neutralization and resolving influenza disease., Methods: We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with the immunostimulating complex Matrix M™. Sixty adults were intramuscularly immunized with two vaccine doses (21 days apart) of 30 μg HA alone or 1.5, 7.5 or 30 μg HA adjuvanted with Matrix M™. Serum H5 HA1-specific antibodies and virus neutralization were determined at days 0, 21, 42, 180 and 360 and long-term memory B cells at day 360 post-vaccination. The binding of the HA specific antibodies was measured by avidity NaSCN-elution ELISA and surface plasmon resonance (SPR)., Results: The H5 HA1-specific IgG response peaked after the second dose (day 42), was dominated by IgG1 and IgG3 and was highest in the adjuvanted vaccine groups. IgG titres correlated significantly with virus neutralization at all time points (Spearman r≥0.66, p<0.0001). By elution ELISA, serum antibody avidity was highest at days 180 and 360 post vaccination and did not correlate with virus neutralization. Long-lasting H5 HA1-specific memory B cells produced high IgG antibody avidity similar to serum IgG., Conclusions: Maturation of serum antibody avidity continued up to day 360 after influenza H5N1 vaccination. Virus neutralization correlated with serum H5 HA1-specific IgG antibody concentrations and not antibody avidity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

T cellular responses play a significant role in mediating protective immune responses against influenza in humans. In the current study, we evaluated the ability of a candidate virosomal H5N1 vaccine adjuvanted with Matrix M(TM) to induce CD4(+) and CD8(+) T cell responses in a phase 1 clinical trial. We vaccinated 60 healthy adult volunteers (at days 0 and 21) with 30 μg haemagglutinin (HA) alone or 1.5, 7.5, or 30 μg HA formulated with Matrix M(TM). To evaluate the T cellular responses, lymphocytes were stimulated in vitro with homologous (A/Vietnam/1194/2004 [H5N1]) and heterologous H5N1 (A/Anhui/1/05 or A/Bar-headed Goose/Qinghai/1A/05) antigens. The antigen-specific cytokine responses were measured by intracellular cytokine staining and by multiplex (Luminex) assays. An increase in CD4(+) Th1 and Th2 cytokines was detected 21 days after the first vaccine dose. No increase in Th cytokine responses was observed after the second dose, although it is possible that the cytokine levels peaked earlier than sampling point at day 42. Formulation with the Matrix M(TM) adjuvant augmented both the homologous and cross-reactive cytokine response. Antigen-specific CD8(+) T cell responses were detected only in a few vaccinated individuals. The concentrations of Th1 and to a lesser extent, Th2 cytokines at 21 days post-vaccination correlated moderately with subsequent days 35 and 180 serological responses as measured by the microneutralisation, haemagglutination inhibition, and single radial hemolysis assays. Results presented here show that the virosomal H5N1 vaccine induced balanced Th1/Th2 cytokine responses and that Matrix M(TM) is a promising adjuvant for future development of candidate pandemic influenza vaccines.

Background: Highly pathogenic avian influenza A/H5N1 virus remains a potential pandemic threat, and it is essential to continue vaccine development against this subtype. A local mucosal immune response in the upper respiratory tract may stop influenza transmission. It is therefore important to develop effective intranasal pandemic influenza vaccines that induce mucosal immunity at the site of viral entry., Objectives: We evaluated the humoral and cellular immune responses of two promising mucosal adjuvants (Chitosan and c-di-GMP) for intranasal influenza H5N1 vaccine in a murine model. Furthermore, we evaluated the concept of co-adjuvanting an experimental adjuvant (c-di-GMP) with chitosan., Methods: BALB/c mice were intranasally immunised with two doses of subunit NIBRG-14 (H5N1) vaccine (7·5, 1·5 or 0·3 μg haemagglutinin (HA) adjuvanted with chitosan (CSN), c-di-GMP or both adjuvants., Results: All adjuvant formulations improved the serum and local antibody responses, with the highest responses observed in the 7·5 μg HA CSN and c-di-GMP-adjuvanted groups. The c-di-GMP provided dose sparing with protective single radial haemolysis (SRH), and haemagglutination inhibition (HI) antibody responses found in the 0·3 μg HA group. CSN elicited a Th2 response, whereas c-di-GMP induced higher frequencies of virus-specific CD4+T cells producing one or more Th1 cytokines (IFN-γ+, IL-2+, TNF-α+). A combination of the two adjuvants demonstrated effectiveness at 7·5 μg HA and triggered a more balanced Th cytokine profile., Conclusion: These data show that combining adjuvants can modulate the Th response and in combination with ongoing studies of adjuvanted intranasal vaccines will dictate the way forward for optimal mucosal influenza vaccines., (© 2012 John Wiley & Sons Ltd.)

Background: We conducted a clinical trial in October 2009 to evaluate the immunogenicity of the AS03-adjuvanted influenza vaccine (pH1N1 vaccine) in health care workers (HCWs). By 2 weeks after vaccination, 97% had protective hemagglutinin inhibition (HI) titers (≥ 40) however, 16% were low responders (LR) and failed to maintain a protective response 90 days after vaccination., Methods: We analyzed the humoral responses (HI, antibody-secreting cell [ASC], and serum immunoglobulin G [IgG]) in 15 LRs and 25 control HCWs. Twelve LRs were revaccinated with the pH1N1 vaccine, and 7 were subsequently vaccinated with the 2010 seasonal trivalent influenza vaccine. We conducted a long-term analysis of the humoral and CD4(+) T-helper (Th) 1 responses., Results: The LRs had a slower HI antibody response than the control HCWs, with protective antibody titers not reached until 2 weeks after vaccination in the majority of the participants. The LRs also had significantly lower IgG ASCs at day 7 and HA1-specific serum IgG responses at day 21, compared with the control HCWs. Revaccination with the pH1N1 vaccine elicited rapid HI antibody, ASC, memory B cell, and multifunctional CD4(+) Th1 cell responses., Conclusion: This study shows that revaccination of low-responding HCWs with the pH1N1 vaccine is required for maintaining long-term protection., Clinical Trials Registration: NCT01003288.