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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).)