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Plasma cytokines are useful indicators of the inflammatory response to vaccination, and can serve as potential biomarkers of the systemic reactogenicity and immunogenicity of vaccines. Measurement of cytokines in urine may represent a non-invasive alternative to the blood-based markers. To evaluate whether urinary cytokine levels can help predict vaccine responses to an AS01

IMPORTANCE: The SARS-CoV-2 variant B.1.1.529 (Omicron) escapes neutralizing antibodies elicited after COVID-19 vaccination, while T-cell responses might be better conserved. It is crucial to assess how a third vaccination modifies these responses, particularly for immunocompromised patients with readily impaired antibody responses. OBJECTIVE: To determine T-cell responses to the Omicron spike protein in anti-CD20–treated patients with multiple sclerosis (MS) before and after a third messenger RNA COVID-19 vaccination. DESIGN, SETTING, AND PARTICIPANTS: In this prospective cohort study conducted from March 2021 to November 2021 at the University Hospital Geneva, adults with MS receiving anti-CD20 treatment (ocrelizumab) were identified by their treating neurologists and enrolled in the study. A total of 20 patients received their third dose of messenger RNA COVID-19 vaccine and were included in this analysis. INTERVENTIONS: Blood sampling before and 1 month after the third vaccine dose. MAIN OUTCOMES AND MEASURES: Quantification of CD4 and CD8 (cytotoxic) T cells specific for the SARS-CoV-2 spike proteins of the vaccine strain as well as the Delta and Omicron variants, comparing frequencies before and after the third vaccine dose. RESULTS: Of 20 included patients, 11 (55%) were male, and the median (IQR) age was 45.8 (37.8-53.3) years. Spike-specific CD4 and CD8 T-cell memory against all variants were maintained in 9 to 12 patients 6 months after their second vaccination, albeit at lower median frequencies against the Delta and Omicron variants compared with the vaccine strain (CD8 T cells: Delta, 83.0%; 95% CI, 73.6-114.5; Omicron, 78.9%; 95% CI, 59.4-100.0; CD4 T cells: Delta, 72.2%; 95% CI, 67.4-90.5; Omicron, 62.5%; 95% CI, 51.0-89.0). A third dose enhanced the number of responders to all variants (11 to 15 patients) and significantly increased CD8 T-cell responses, but the frequencies of Omicron-specific CD8 T cells remained 71.1% (95% CI, 41.6-96.2) of the responses specific to the vaccine strain. CONCLUSIONS AND RELEVANCE: In this cohort study of patients with MS treated with ocrelizumab, there were robust T-cell responses recognizing spike proteins from the Delta and Omicron variants, suggesting that COVID-19 vaccination in patients taking B-cell–depleting drugs may protect them against serious complications from COVID-19 infection. T-cell response rates increased after the third dose, demonstrating the importance of a booster dose for this population.

A recurrent question is whether transient reactions to vaccines translate into better immune responses. Using clinical data from 2 large phase 3 studies of the recombinant zoster vaccine, we observed a small but statistically significant association between the intensity of a frequent side effect (pain) after vaccination and immune responses to vaccination. However, despite the statistical correlation, the impact on the immune response is so small, and the immune response in individuals without pain already sufficient, that pain cannot be a surrogate marker for an appropriate immune response. Reactogenicity cannot be used to predict immunity after vaccination.

The candidate Adjuvant System AS37 contains a synthetic toll-like receptor agonist (TLR7a) adsorbed to alum. In a phase I study (NCT02639351), healthy adults were randomised to receive one dose of licensed alum-adjuvanted meningococcal serogroup C (MenC-CRM

Emerging SARS-CoV-2 variants of concern/interest (VOC/VOI) raise questions about effectiveness of neutralizing antibodies derived from infection or vaccination. As the population immunity to SARS-CoV-2 has become more complex due to prior infection and/or vaccination, understanding the antigenic relationship between variants is needed. Here, we have assessed in total 104 blood specimens from convalescent individuals after infection with early-pandemic SARS-CoV-2 (pre-VOC) or with Alpha, Beta, Gamma or Delta, post-vaccination after double-dose mRNA-vaccination and break through infections due to Delta or Omicron. Neutralization against seven authentic SARS-CoV-2 isolates (B.1, Alpha, Beta, Gamma, Delta, Zeta, Omicron) was assessed by plaque-reduction neutralization assay. We found highest neutralization titers against the homologous (previously infecting) variant, with lower neutralization efficiency against heterologous variants. Significant loss of neutralization for Omicron was observed but to a varying degree depending on previously infecting variant (23.0-fold in Beta-convalescence up to 56.1-fold in Alpha-convalescence), suggesting that infection-derived immunity varies, but independent of the infecting variant is only poorly protective against Omicron. Of note, Zeta VOI showed also pronounced escape from neutralization of up to 28.2-fold in Alpha convalescent samples. Antigenic mapping reveals both Zeta and Omicron as separate antigenic clusters. Double dose vaccination showed robust neutralization for Alpha, Beta, Gamma, Delta and Zeta, with fold-change reduction of only 2.8 (for Alpha) up to 6.9 (for Beta). Escape from neutralization for Zeta was largely restored in vaccinated individuals, while Omicron still showed a loss of neutralization of 85.7-fold compared to pre-VOC SARS-CoV-2. Combined immunity from infection followed by vaccination or vaccine breakthrough infection showed highest titers and most robust neutralization for heterologous variants. Breakthrough infection with Delta showed only 12.5-fold reduced neutralization for Omicron, while breakthrough infection with Omicron showed only a 1.5-fold loss for Delta, suggests that infection with antigenically different variants can boost immunity for antigens closer to the vaccine strain. Antigenic cartography showed also a tendency towards broader neutralizing capacity for heterologous variants. We conclude that the complexity of background immunity needs to be taken into account when assessing new VOCs. Development towards separate serotypes such as Zeta was already observed before Omicron emergence, thus other factors than just immune escape must contribute to Omicrons rapid dominance. However, combined infection/vaccination immunity could ultimately lead to broad neutralizing capacity also against non-homologous variants.

Differences in innate immune ‘imprinting’ between vaccine adjuvants may mediate dissimilar effects on the quantity/quality of persisting adaptive responses. We compared antibody avidity maturation, antibody/memory B cell/CD4+ T cell response durability, and recall responses to non-adjuvanted fractional-dose antigen administered 1-year post-immunization (Day [D]360), between hepatitis B vaccines containing Adjuvant System (AS)01B, AS01E, AS03, AS04, or Alum (NCT00805389). Both the antibody and B cell levels ranked similarly (AS01B/E/AS03 > AS04 > Alum) at peak response, at D360, and following their increases post-antigen recall (D390). Proportions of high-avidity antibodies increased post-dose 2 across all groups and persisted at D360, but avidity maturation appeared to be more strongly promoted by AS vs. Alum. Post-antigen recall, frequencies of subjects with high-avidity antibodies increased only markedly in the AS groups. Among the AS, total antibody responses were lowest for AS04. However, proportions of high-avidity antibodies were similar between groups, suggesting that MPL in AS04 contributes to avidity maturation. Specific combinations of immunoenhancers in the AS, regardless of their individual nature, increase antibody persistence and avidity maturation., SCOPUS: ar.j, info:eu-repo/semantics/published

Children are unique in the context of the COVID-19 pandemic. Overall, SARS-CoV-2 has a lower medical impact in children as compared to adults. A higher proportion of children than adults remain asymptomatic following SARS-CoV-2 infection and severe disease and death are also less common. This relative resistance contrasts with the high susceptibility of children to other respiratory tract infections. The mechanisms involved remain incompletely understood but could include the rapid development of a robust innate immune response. On the other hand, children develop a unique and severe complication, named multisystem inflammatory syndrome in children, several weeks after the onset of symptoms. Although children play an important role in the transmission of many pathogens, their contribution to the transmission of SARS-CoV-2 appears lower than that of adults. These unique aspects of COVID-19 in children must be considered in the benefit–risk analysis of vaccination. Several COVID-19 vaccines have been authorized for emergency use in adolescents and clinical studies are ongoing in children. As the vaccination of adolescents is rolled out in several countries, we shall learn about the impact of this strategy on the health of children and on transmission within communities., SCOPUS: re.j, info:eu-repo/semantics/published

Patients treated with anti-CD20 therapy are particularly at risk of developing severe COVID-19, however little is known regarding COVID-19 vaccine effectiveness in this population. This study assesses humoral and T-cell responses to mRNA-based COVID-19 vaccines in patients treated with rituximab for rheumatic diseases or ocrelizumab for multiple sclerosis (n=37), compared to immunocompetent individuals (n=22). SARS-CoV-2-specific antibodies were detectable in only 69.4% of patients and at levels that were significantly lower compared to controls who all seroconverted. In contrast to antibodies, Spike (S)-specific CD4+ T cells were equally detected in immunocompetent and anti-CD20 treated patients (85-90%) and mostly of a Th1 phenotype. Response rates of S-specific CD8+T cells were higher in ocrelizumab (96.2%) and rituximab-treated patients (81.8%) as compared to controls (66.7%). Vaccine-specific CD4+and CD8+T cells were polyfunctional but expressed more IL-2 in patients than in controls. In summary, our study suggests that patients on anti-CD20 treatment are able to mount potent T-cell responses to mRNA COVID-19 vaccines, despite impaired humoral responses. This could play an important role in the prevention of severe COVID-19.

To evaluate longitudinally the persistence of humoral immunity for up to 6 months in a cohort of hospital employees with mild coronavirus disease 2019 (COVID-19).We measured anti-RBD (receptor binding domain of viral spike protein), anti-N (viral nucleoprotein) and neutralizing antibodies at 1, 3 and 6 months after mostly mild COVID-19 in 200 hospital workers using commercial ELISAs and a surrogate virus neutralization assay.Antibodies specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persisted in all participants for up to 6 months. Anti-RBD geometric mean concentrations (GMCs) progressively increased between months 1 (74.2 U/mL, 95%CI: 62.7-87.8), 3 (103.2 U/mL, 95%CI: 87.9-121.2; p 0.001), and 6 (123.3 U/mL, 95%CI: 103.4-147.0; p 0.001) in the whole cohort. Anti-N antibodies were detectable in97% at all times. Neutralizing antibodies were detectable in 99.5% of participants (195/196) at 6 months post infection. Their GMC progressively decreased between months 1 (20.1 AU/mL, 95%CI: 16.9-24.0), 3 (15.2 AU/mL, 95%CI: 13.2-17.6; p 0.001) and 6 (9.4 AU/mL, 95%CI: 7.7-11.4; p 0.001). RBD-ACE2-inhibiting antibody titres and anti-RBD antibody concentrations strongly correlated at each timepoint (all r 0.86, p 0.001). Disease severity was associated with higher initial anti-RBD and RBD-ACE2-inhibiting antibody titres, but not with their kinetics.Neutralizing antibodies persisted at 6 months in almost all participants, indicating more durability than initially feared. Anti-RBD antibodies persisted better and even increased over time, possibly related to the preferential detection of progressively higher-affinity antibodies.

Background SARS-CoV-2 infection leads to high viral loads in the upper respiratory tract that may be determinant in virus dissemination. The extent of intranasal antiviral response in relation to symptoms is unknown. Understanding how local innate responses control virus is key in the development of therapeutic approaches. Methods SARS-CoV-2-infected patients were enrolled in an observational study conducted at the Geneva University Hospitals, Switzerland, investigating virological and immunological characteristics. Nasal wash and serum specimens from a subset of patients were collected to measure viral load, IgA specific for the S1 domain of the spike protein, and a cytokine panel at different time points after infection; cytokine levels were analyzed in relation to symptoms. Results Samples from 13 SARS-CoV-2-infected patients and six controls were analyzed. We found an increase in CXCL10 and IL-6, whose levels remained elevated for up to 3 weeks after symptom onset. SARS-CoV-2 infection also induced CCL2 and GM-CSF, suggesting local recruitment and activation of myeloid cells. Local cytokine levels correlated with viral load but not with serum cytokine levels, nor with specific symptoms, including anosmia. Some patients had S1-specific IgA in the nasal cavity while almost none had IgG. Conclusion The nasal epithelium is an active site of cytokine response against SARS-CoV-2 that can last more than 2 weeks; in this mild COVID-19 cohort, anosmia was not associated with increases in any locally produced cytokines.

Objectives To report a case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection 6 months after the first infection in a young healthy female physician. Both episodes led to mild coronavirus disease 2019 (COVID-19). Methods SARS-CoV-2 infections were detected by real-time reverse transcriptase PCR (RT-PCR) on nasopharyngeal specimens. Reinfection was confirmed by whole-genome sequencing. Kinetics of total anti-S receptor binding domain immunoglobulins (Ig anti–S RBD), anti-nucleoprotein (anti-N) and neutralizing antibodies were determined in serial serum samples retrieved during both infection episodes. Memory B-cell responses were assessed at day 12 after reinfection. Results Whole-genome sequencing identified two different SARS-CoV-2 genomes both belonging to clade 20A, with only one nonsynonymous mutation in the spike protein and clustered with viruses circulating in Geneva (Switzerland) at the time of each of the corresponding episodes. Seroconversion was documented with low levels of total Ig anti–S RBD and anti-N antibodies at 1 month after the first infection, whereas neutralizing antibodies quickly declined after the first episode and then were boosted by the reinfection, with high titres detectable 4 days after symptom onset. A strong memory B-cell response was detected at day 12 after onset of symptoms during reinfection, indicating that the first episode elicited cellular memory responses. Conclusions Rapid decline of neutralizing antibodies may put medical personnel at risk of reinfection, as shown in this case. However, reinfection leads to a significant boosting of previous immune responses. Larger cohorts of reinfected subjects with detailed descriptions of their immune responses are needed to define correlates of protection and their duration after infection.

The current routine use of adjuvants in human vaccines provides a strong incentive to increase our understanding of how adjuvants differ in their ability to stimulate innate immunity and consequently enhance vaccine immunogenicity. Here, we evaluated gene expression profiles in cells from whole blood elicited in naive subjects receiving the hepatitis B surface antigen formulated with different adjuvants. We identified a core innate gene signature emerging 1 day after the second vaccination and that was shared by the recipients of vaccines formulated with adjuvant systems AS01B, AS01E, or AS03. This core signature associated with the magnitude of the hepatitis B surface-specific antibody response and was characterized by positive regulation of genes associated with interferon-related responses or the innate cell compartment and by negative regulation of natural killer cell-associated genes. Analysis at the individual subject level revealed that the higher immunogenicity of AS01B-adjuvanted vaccine was linked to its ability to induce this signature in most vaccinees even after the first vaccination. Therefore, our data suggest that adjuvanticity is not strictly defined by the nature of the receptors or signaling pathways it activates but by the ability of the adjuvant to consistently induce a core inflammatory signature across individuals.

BackgroundViral shedding patterns and its correlation with the immune responses of mildly symptomatic COVID-19 patients are still poorly characterized.MethodsWe enrolled the first five COVID-19 patients quarantined in our institution; none received immunomodulatory treatment. We monitored shedding of viral RNA and infectious virus by RT-PCR and cell culture from the upper respiratory tract, and characterized the kinetics of systemic innate and adaptive immune responses.ResultsDespite mild clinical disease, high viral loads and shedding of infectious virus were observed from the respiratory tract, with isolation of infectious virus and prolonged positivity by PCR up to day 7 and 19 post onset of symptoms, respectively. Robust innate responses characterized by an increase in activated CD14+CD16+ monocytes and cytokine responses were observed as early as 2 days after symptoms onset. Cellular and humoral SARS-CoV-2 specific adaptive responses were detectable in all patients.ConclusionInfectious virus shedding was limited to the first week of symptom onset in mild cases. A strong innate response, characterized by the mobilization of activated monocytes during the first days of infection, as well as SARS-CoV-2 specific antibodies were detectable, even in patients with mild disease.SummaryWe describe viral and immune profiles of the first five SARS-CoV-2 patients in our institution, showing high viral loads and infectious viral shedding in early acute disease. Mild patients mount an innate response sufficient for viral control and specific immunity.

A novel coronavirus (CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 in Wuhan, China and has since spread as a global pandemic. Safe and effective vaccines are thus urgently needed to reduce the significant morbidity and mortality of Coronavirus Disease 2019 (COVID-19) disease and ease the major economic impact. There has been an unprecedented rapid response by vaccine developers with now over one hundred vaccine candidates in development and at least six having reached clinical trials. However, a major challenge during rapid development is to avoid safety issues both by thoughtful vaccine design and by thorough evaluation in a timely manner. A syndrome of “disease enhancement” has been reported in the past for a few viral vaccines where those immunized suffered increased severity or death when they later encountered the virus or were found to have an increased frequency of infection. Animal models allowed scientists to determine the underlying mechanism for the former in the case of Respiratory Syncytial virus (RSV) vaccine and have been utilized to design and screen new RSV vaccine candidates. Because some Middle East respiratory syndrome (MERS) and SARS-CoV-1 vaccines have shown evidence of disease enhancement in some animal models, this is a particular concern for SARS-CoV-2 vaccines. To address this challenge, the Coalition for Epidemic Preparedness Innovations (CEPI) and the Brighton Collaboration (BC) Safety Platform for Emergency vACcines (SPEAC) convened a scientific working meeting on March 12 and 13, 2020 of experts in the field of vaccine immunology and coronaviruses to consider what vaccine designs could reduce safety concerns and how animal models and immunological assessments in early clinical trials can help to assess the risk. This report summarizes the evidence presented and provides considerations for safety assessment of COVID-19 vaccine candidates in accelerated vaccine development.

International audience; The 3-O-desacyl-4'-monophosphoryl lipid A (MPL) activates immunity through Toll-like receptor 4 (TLR4) signaling. The Adjuvant System AS01 contains MPL and is used in the candidate malaria vaccine and the licensed zoster vaccine. Recent studies reported that AS01 adjuvant activity depends on a transient inflammation at the site of vaccination, but the role of stromal or structural cells in the adjuvant effect is unknown. We investigated this question in mouse models by assessing the role of TLR4 on hematopoietic versus resident structural cells during immunization with AS01-adjuvanted vaccines. We first established that TLR4-deficient animals had a reduced immune response to an AS01-adjuvanted vaccine. Using bone marrow chimera, we consistently found that Tlr4 expression in radio-sensitive cells, i.e., hematopoietic cells, was required for an optimal adjuvant effect on antibody and T-cell responses. At day 1 after injection, the pro-inflammatory reaction at the site of injection was strongly dependent on TLR4 signaling in hematopoietic cells. Similarly, activation of dendritic cells in muscle-draining lymph nodes was strictly associated with the radio-sensitive cells expressing Tlr4. Altogether, these data suggest that MPL-mediated TLR4-signaling in hematopoietic cells is critical in the mode of action of AS01.

Tissue residency is considered a defining feature of the innate lymphoid cell (ILC) populations located within mucosal and adipose tissues. ILCs are also present within all lymphoid tissues, but whether ILCs migrate between lymphoid and nonlymphoid sites and in what context is poorly understood. To determine whether migratory ILCs exist within peripheral lymph nodes (LNs), we labeled all cells within the brachial LN (bLN) of transgenic mice expressing a photoconvertible fluorescent protein by direct exposure to light. Tracking of cellular changes in the labeled LN revealed the gradual migration of new ILCs into the tissue, balanced by egress of ILCs dependent on sphingosine-1-phosphate receptors. Most of the migratory ILCs were ILC1s, entering LNs directly from the circulation in a CD62L- and CCR7-dependent manner and thus behaving like conventional natural killer (cNK) cells. Upon egress, both ILC1s and cNK cells were found to recirculate through peripheral LNs. A distinct population of migratory ILC2s were detected in the LN, but most of the ILC3s were tissue resident. Functionally, both migratory and resident ILC1s within LNs were able to rapidly produce IFN-γ to support the generation of robust TH1 T cell responses after immunization. Thus, migratory and resident ILC populations exist within peripheral LNs, with ILC1s, akin to cNK cells, able to traffic into these tissues where they can contribute to the initiation of adaptive immunity.

Adjuvants are used to improve vaccine immunogenicity and efficacy by enhancing antigen presentation to antigen-specific immune cells with the aim to confer long-term protection against targeted pathogens. Adjuvants have been used in vaccines for more than 90 years. Combinations of immunostimulatory molecules, such as in the Adjuvant System AS01, have opened the way to the development of new or improved vaccines. Areas covered: AS01 is a liposome-based vaccine adjuvant system containing two immunostimulants: 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and the saponin QS-21. Here we describe studies investigating the mode of action of AS01, and consider the role of AS01 in enhancing specific immune responses to the antigen for selected candidate vaccines targeting malaria and herpes zoster. The effects of AS01 are rapid and transient, being localized to the injected muscle and draining lymph node. AS01 is efficient at promoting CD4

Immunogenicity and safety of different adjuvants combined with a model antigen (HBsAg) were compared. Healthy HBV-naïve adults were randomized to receive HBs adjuvanted with alum or Adjuvant Systems AS01B, AS01E, AS03A or AS04 at Days 0 and 30. Different frequencies of HBs-specific CD4. + T cells 14 days post dose 2 but similar polyfunctionality profiles were induced by the different adjuvants with frequencies significantly higher in the AS01B and AS01E groups than in the other groups. Antibody concentrations 30 days post-dose 2 were significantly higher in AS01B, AS01E and AS03A than in other groups. Limited correlations were observed between HBs-specific CD4. + T cell and antibody responses. Injection site pain was the most common solicited local symptom and was more frequent in AS groups than in alum group. Different adjuvants formulated with the same antigen induced different adaptive immune responses and reactogenicity patterns in healthy naïve adults. The results summary for this study (GSK study number 112115 - NCT, SCOPUS: ar.j, info:eu-repo/semantics/published

The oil-in-water emulsion Adjuvant System 03 (AS03) is one of the few adjuvants used in licensed vaccines. Previous work indicates that AS03 induces a local and transient inflammatory response that contributes to its adjuvant effect. However, the molecular mechanisms involved in its immunostimulatory properties are ill-defined. Upon intramuscular injection in mice, AS03 elicited a rapid and transient downregulation of lipid metabolism-related genes in the draining lymph node. In vitro, these modifications were associated with profound changes in lipid composition, alteration of endoplasmic reticulum (ER) morphology and activation of the unfolded protein response pathway. In vivo, treatment with a chemical chaperone or deletion of the ER stress sensor kinase IRE1α in myeloid cells decreased AS03-induced cytokine production and its capacity to elicit high affinity antigen-specific antibodies. In summary, our results indicate that IRE1α is a sensor for the metabolic changes induced by AS03 in monocytic cells and may constitute a canonical pathway that could be exploited for the design of novel vaccine adjuvants., SCOPUS: ar.j, info:eu-repo/semantics/published

After decades of slow progress, the last years have seen a rapid acceleration of the development of adjuvanted vaccines which have lately been approved for human use. These adjuvants consist of different components, e.g. aluminium salts, emulsions such as MF59 and AS03, Toll-like receptor (TLR) agonists (CpG ormonophosphoryl lipid A (MPL) adsorbed on aluminium salts as in AS04) or combination of immunopotentiators (QS-21 and MPL in AS01). Despite their distinctive features, most of these adjuvants share some key characteristics. For example, they induce early activation (although at different levels) of innate immunity which then translates into higher antibody and cellular responses to the vaccine antigens. In addition, most of these adjuvants (e.g. MF59, AS03, AS04) clearly induce a wider breadth of adaptive responses able to confer protection against, for example, heterovariants of the influenza viruses (MF59, AS03) or against human papillomavirus strains not contained in the vaccine (AS04). Finally, the use of some of these adjuvants has contributed to significantly enhance the immune response and the efficacy and effectiveness of vaccines in the elderly who experience a waning of the immune responsiveness to infection and vaccination, as shown for MF59- or AS03-adjuvanted influenza vaccines and AS01-adjuvanted herpes zoster vaccine. These results, together with the track record of acceptable safety profiles of the adjuvanted vaccines, pave the way for the development of novel vaccines at the extremes of age and against infections with a high toll of morbidity and mortality. Here, we review the mechanisms associated with the performance of those adjuvanted vaccines in animal models and in humans through recent advances in systems vaccinology and biomarker discovery. We also provide some perspectives on remaining knowledge gaps but also on opportunities that could accelerate the development of new vaccines.

Pandemic-influenza vaccines containing split-inactivated-virus antigen have been formulated with the immunostimulatory Adjuvant System AS03 to enhance the antigen immunogenicity and reduce antigen content per dose. AS03 is an oil-in-water emulsion containing α-tocopherol, squalene and polysorbate 80. To support the clinical development of AS03-adjuvanted pandemic-influenza vaccines, the local and systemic toxicity of test articles containing split-influenza A(H5N1) and/or AS03 were evaluated after 3-4 intramuscular (i.m.) injections in rabbits. Treatment-related effects were restricted to mild inflammatory responses and were induced primarily by the test articles containing AS03. The injection-site inflammation was mild at 3 days, and minimal at 4 weeks after the last injection; and was reflected by signs of activation in the draining lymph nodes and by systemic effects in the blood including a transient increase of neutrophils. In addition, a study in mice explored the biodistribution of A(H5N1) vaccines or AS03 through radiolabelling the antigen or constituents of AS03 prior to injection. In this evaluation, 57-73% of AS03's principal constituents had cleared from the injection site 3 days after injection, and their different clearance kinetics were suggestive of AS03's dissociation. All these AS03 constituents entered into the draining lymph nodes within 30 min after injection. In conclusion, the administration of repeated doses of the H5N1/AS03 vaccine was well tolerated in the rabbit, and was primarily associated with transient mild inflammation at the injection site and draining lymph nodes. The biodistribution kinetics of AS03 constituents in the mouse were consistent with AS03 inducing this pattern of inflammation. Copyright © 2015 John Wiley & Sons, Ltd.

Combining immunostimulants in adjuvants can improve the quality of the immune response to vaccines. Here, we report a unique mechanism of molecular and cellular synergy between a TLR4 ligand, 3-O-desacyl-4’-monophosphoryl lipid A (MPL), and a saponin, QS-21, the constituents of the Adjuvant System AS01. AS01 is part of the malaria and herpes zoster vaccine candidates that have demonstrated efficacy in phase III studies. Hours after injection of AS01-adjuvanted vaccine, resident cells, such as NK cells and CD8+ T cells, release IFNγ in the lymph node draining the injection site. This effect results from MPL and QS-21 synergy and is controlled by macrophages, IL-12 and IL-18. Depletion strategies showed that this early IFNγ production was essential for the activation of dendritic cells and the development of Th1 immunity by AS01-adjuvanted vaccine. A similar activation was observed in the lymph node of AS01-injected macaques as well as in the blood of individuals receiving the malaria RTS,S vaccine. This mechanism, previously described for infections, illustrates how adjuvants trigger naturally occurring pathways to improve the efficacy of vaccines.

Saponins represent a promising class of vaccine adjuvant. Together with the TLR4-ligand MPL, QS-21 is part of the Adjuvant System AS01, a key component of the malaria and zoster candidate vaccines that display demonstrated clinical efficacy. However, the mechanism of action of QS-21 in this liposomal formulation is poorly understood. Upon intra-muscular immunisation, we observed that QS-21 rapidly accumulated in CD169(+) resident macrophages of the draining lymph node where it elicited a local innate immune response. Depletion of these cells abrogated QS-21-mediated innate cell recruitment to the lymph node, dendritic cell (DC) phenotypic maturation as well as the adjuvant effect on T-cell and antibody responses to co-administered antigens. DCs rather than lymph node-resident macrophages were directly involved in T-cell priming by QS-21, as revealed by the decrease in antigen-specific T-cell response in Batf3(-/-) mice. Further analysis showed that the adjuvant effect of QS-21 depended on the integration of Caspase-1 and MyD88 pathways, at least in part through the local release of HMGB1. Taken together, this work unravels the key role of lymph node sentinel macrophage in controlling the adjuvant effect of a molecule proven to improve vaccine response in humans., SCOPUS: ar.j, info:eu-repo/semantics/published

The adjuvant properties of the saponin QS-21 have been known for decades. It is a component of the Adjuvant System AS01 that is used in several vaccine candidates. QS-21 strongly potentiates both cellular and humoral immune responses to purified antigens, yet how it activates immune cells is largely unknown. Here, we report that QS-21 directly activated human monocyte-derived dendritic cells (moDCs) and promoted a pro-inflammatory transcriptional program. Cholesterol-dependent QS-21 endocytosis followed by lysosomal destabilization and Syk kinase activation were prerequisites for this response. Cathepsin B, a lysosomal cysteine protease, was essential for moDC activation in vitro and contributed to the adjuvant effects of QS-21 in vivo. Collectively, these findings provide new insights into the pathways involved in the direct activation of antigen-presenting cells by a clinically relevant QS-21 formulation., SCOPUS: ar.j, info:eu-repo/semantics/published

Pandemic-influenza vaccines containing split-inactivated-virus antigen have been formulated with the immunostimulatory Adjuvant System AS03 to enhance the antigen immunogenicity and reduce antigen content per dose. AS03 is an oil-in-water emulsion containing α-tocopherol, squalene and polysorbate 80. To support the clinical development of AS03-adjuvanted pandemic-influenza vaccines, the local and systemic toxicity of test articles containing split-influenza A(H5N1) and/or AS03 were evaluated after 3-4 intramuscular (i.m.) injections in rabbits. Treatment-related effects were restricted to mild inflammatory responses and were induced primarily by the test articles containing AS03. The injection-site inflammation was mild at 3 days, and minimal at 4 weeks after the last injection; and was reflected by signs of activation in the draining lymph nodes and by systemic effects in the blood including a transient increase of neutrophils. In addition, a study in mice explored the biodistribution of A(H5N1) vaccines or AS03 through radiolabelling the antigen or constituents of AS03 prior to injection. In this evaluation, 57-73% of AS03's principal constituents had cleared from the injection site 3 days after injection, and their different clearance kinetics were suggestive of AS03's dissociation. All these AS03 constituents entered into the draining lymph nodes within 30 min after injection. In conclusion, the administration of repeated doses of the H5N1/AS03 vaccine was well tolerated in the rabbit, and was primarily associated with transient mild inflammation at the injection site and draining lymph nodes. The biodistribution kinetics of AS03 constituents in the mouse were consistent with AS03 inducing this pattern of inflammation.