Alum/Toll-Like Receptor 7 Adjuvant Enhances the Expansion of Memory B Cell Compartment Within the Draining Lymph Node

Vaccination is one of the most cost-effective health interventions and, with the exception of water sanitization, no other action has had such a major effect in mortality reduction. Combined with other approaches, such as clean water, better hygiene, and health education, vaccination contributed to prevent millions of cases of deaths among children under 5 years of age. New or improved vaccines are needed to fight some vaccine-preventable diseases that are still a threat for the public health globally, as reported also in the Global Vaccine Action Plan (GVAP) endorsed by the World Health Assembly in 2012. Adjuvants are substances that enhance the effectiveness of vaccination, but despite their critical role for the development of novel vaccines, very few of them are approved for use in humans. Aluminum hydroxide (Alum) is the most common adjuvant used in vaccines administered in millions of doses around the world to prevent several dangerous diseases. The development of an improved version of Alum can help to design and produce new or better vaccines. Alum/toll-like receptor (TLR)7 is a novel Alum-based adjuvant, currently in phase I clinical development, formed by the attachment of a benzonaphthyridine compound, TLR7 agonist, to Alum. In preclinical studies, Alum/TLR7 showed a superior adjuvant capacity, compared to Alum, in several disease models, such as meningococcal meningitis, anthrax, staphylococcus infections. None of these studies reported the effect of Alum/TLR7 on the generation of the B cell memory compartment, despite this is a critical aspect to achieve a better immunization. In this study, we show, for the first time, that, compared to Alum, Alum/TLR7 enhances the expansion of the memory B cell compartment within the draining lymph node (LN) as result of intranodal sustained proliferation of antigen-engaged B cells and/or accumulation of memory B cells. In addition, we observed that Alum/TLR7 induces a recruitment of naïve antigen-specific B cells within the draining LN that may help to sustain the germinal center reaction. Our data further support Alum/TLR7 as a new promising adjuvant, which might contribute to meet the expectations of the GVAP for 2020 and beyond

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<p>Toll-like receptors (TLRs) play a key role in the activation of innate immune cells, in which their engagement leads to production of cytokines and co-stimulatory molecules. TLRs signaling requires recruitment of toll/IL-1R (TIR) domain-containing adaptors, such as MyD88 and/or TRIF, and leads to activation of several transcription factors, such as NF-κB, the AP1 complex, and various members of the interferon regulatory factor (IRF) family, which in turn results in triggering of several cellular functions associated with these receptors. A role for Src family kinases (SFKs) in this signaling pathway has also been established. Our work and that of others have shown that this type of kinases is activated following engagement of several TLRs, and that this event is essential for the initiation of specific downstream cellular response. In particular, we have previously demonstrated that activation of SFKs is required for balanced production of pro-inflammatory cytokines by monocyte-derived dendritic cells after stimulation with R848, an agonist of human TLRs 7/8. We also showed that TLR7/8 triggering leads to an increase in interferon regulatory factor 1 (IRF-1) protein levels and that this effect is abolished by inhibition of SFKs, suggesting a critical role of these kinases in IRF-1 regulation. In this study, we first confirmed the key role of SFKs in TLR7/8 signaling for cytokine production and accumulation of IRF-1 protein in monocytes and in B lymphocytes, two other type of antigen-presenting cells. Then, we demonstrate that TLR7 triggering leads to an increase of K63-linked ubiquitination of IRF-1, which is prevented by SFKs inhibition, suggesting a key role of these kinases in posttranslational regulation of IRF-1 in the immune cells. In order to understand the mechanism that links SFKs activation to IRF-1 K63-linked ubiquitination, we examined SFKs and IRF-1 possible interactors and proved that activation of SFKs is necessary for their interaction with TNFR-associated factor 6 (TRAF6) and promotes the recruitment of both cIAP2 and IRF-1 by TRAF6. Collectively, our data demonstrate that TLR7/8 engagement leads to the formation of a complex that allows the interaction of cIAP2 and IRF-1 resulting in IRF-1 K63-linked ubiquitination, and that active SFKs are required for this process.</p

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<p>Vaccination is one of the most cost-effective health interventions and, with the exception of water sanitization, no other action has had such a major effect in mortality reduction. Combined with other approaches, such as clean water, better hygiene, and health education, vaccination contributed to prevent millions of cases of deaths among children under 5 years of age. New or improved vaccines are needed to fight some vaccine-preventable diseases that are still a threat for the public health globally, as reported also in the Global Vaccine Action Plan (GVAP) endorsed by the World Health Assembly in 2012. Adjuvants are substances that enhance the effectiveness of vaccination, but despite their critical role for the development of novel vaccines, very few of them are approved for use in humans. Aluminum hydroxide (Alum) is the most common adjuvant used in vaccines administered in millions of doses around the world to prevent several dangerous diseases. The development of an improved version of Alum can help to design and produce new or better vaccines. Alum/toll-like receptor (TLR)7 is a novel Alum-based adjuvant, currently in phase I clinical development, formed by the attachment of a benzonaphthyridine compound, TLR7 agonist, to Alum. In preclinical studies, Alum/TLR7 showed a superior adjuvant capacity, compared to Alum, in several disease models, such as meningococcal meningitis, anthrax, staphylococcus infections. None of these studies reported the effect of Alum/TLR7 on the generation of the B cell memory compartment, despite this is a critical aspect to achieve a better immunization. In this study, we show, for the first time, that, compared to Alum, Alum/TLR7 enhances the expansion of the memory B cell compartment within the draining lymph node (LN) as result of intranodal sustained proliferation of antigen-engaged B cells and/or accumulation of memory B cells. In addition, we observed that Alum/TLR7 induces a recruitment of naïve antigen-specific B cells within the draining LN that may help to sustain the germinal center reaction. Our data further support Alum/TLR7 as a new promising adjuvant, which might contribute to meet the expectations of the GVAP for 2020 and beyond.</p

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<p>Vaccination is one of the most cost-effective health interventions and, with the exception of water sanitization, no other action has had such a major effect in mortality reduction. Combined with other approaches, such as clean water, better hygiene, and health education, vaccination contributed to prevent millions of cases of deaths among children under 5 years of age. New or improved vaccines are needed to fight some vaccine-preventable diseases that are still a threat for the public health globally, as reported also in the Global Vaccine Action Plan (GVAP) endorsed by the World Health Assembly in 2012. Adjuvants are substances that enhance the effectiveness of vaccination, but despite their critical role for the development of novel vaccines, very few of them are approved for use in humans. Aluminum hydroxide (Alum) is the most common adjuvant used in vaccines administered in millions of doses around the world to prevent several dangerous diseases. The development of an improved version of Alum can help to design and produce new or better vaccines. Alum/toll-like receptor (TLR)7 is a novel Alum-based adjuvant, currently in phase I clinical development, formed by the attachment of a benzonaphthyridine compound, TLR7 agonist, to Alum. In preclinical studies, Alum/TLR7 showed a superior adjuvant capacity, compared to Alum, in several disease models, such as meningococcal meningitis, anthrax, staphylococcus infections. None of these studies reported the effect of Alum/TLR7 on the generation of the B cell memory compartment, despite this is a critical aspect to achieve a better immunization. In this study, we show, for the first time, that, compared to Alum, Alum/TLR7 enhances the expansion of the memory B cell compartment within the draining lymph node (LN) as result of intranodal sustained proliferation of antigen-engaged B cells and/or accumulation of memory B cells. In addition, we observed that Alum/TLR7 induces a recruitment of naïve antigen-specific B cells within the draining LN that may help to sustain the germinal center reaction. Our data further support Alum/TLR7 as a new promising adjuvant, which might contribute to meet the expectations of the GVAP for 2020 and beyond.</p

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<p>Vaccination is one of the most cost-effective health interventions and, with the exception of water sanitization, no other action has had such a major effect in mortality reduction. Combined with other approaches, such as clean water, better hygiene, and health education, vaccination contributed to prevent millions of cases of deaths among children under 5 years of age. New or improved vaccines are needed to fight some vaccine-preventable diseases that are still a threat for the public health globally, as reported also in the Global Vaccine Action Plan (GVAP) endorsed by the World Health Assembly in 2012. Adjuvants are substances that enhance the effectiveness of vaccination, but despite their critical role for the development of novel vaccines, very few of them are approved for use in humans. Aluminum hydroxide (Alum) is the most common adjuvant used in vaccines administered in millions of doses around the world to prevent several dangerous diseases. The development of an improved version of Alum can help to design and produce new or better vaccines. Alum/toll-like receptor (TLR)7 is a novel Alum-based adjuvant, currently in phase I clinical development, formed by the attachment of a benzonaphthyridine compound, TLR7 agonist, to Alum. In preclinical studies, Alum/TLR7 showed a superior adjuvant capacity, compared to Alum, in several disease models, such as meningococcal meningitis, anthrax, staphylococcus infections. None of these studies reported the effect of Alum/TLR7 on the generation of the B cell memory compartment, despite this is a critical aspect to achieve a better immunization. In this study, we show, for the first time, that, compared to Alum, Alum/TLR7 enhances the expansion of the memory B cell compartment within the draining lymph node (LN) as result of intranodal sustained proliferation of antigen-engaged B cells and/or accumulation of memory B cells. In addition, we observed that Alum/TLR7 induces a recruitment of naïve antigen-specific B cells within the draining LN that may help to sustain the germinal center reaction. Our data further support Alum/TLR7 as a new promising adjuvant, which might contribute to meet the expectations of the GVAP for 2020 and beyond.</p

The respiratory syncytial virus (RSV) prefusion F‐protein functional antibody repertoire in adult healthy donors

Abstract Respiratory syncytial virus (RSV) is the leading cause of death from lower respiratory tract infection in infants and children, and is responsible for considerable morbidity and mortality in older adults. Vaccines for pregnant women and elderly which are in phase III clinical studies target people with pre‐existing natural immunity against RSV. To investigate the background immunity which will be impacted by vaccination, we single cell‐sorted human memory B cells and dissected functional and genetic features of neutralizing antibodies (nAbs) induced by natural infection. Most nAbs recognized both the prefusion and postfusion conformations of the RSV F‐protein (cross‐binders) while a smaller fraction bound exclusively to the prefusion conformation. Cross‐binder nAbs used a wide array of gene rearrangements, while preF‐binder nAbs derived mostly from the expansion of B‐cell clonotypes from the IGHV1 germline. This latter class of nAbs recognizes an epitope located between Site Ø, Site II, and Site V on the F‐protein, identifying an important site of pathogen vulnerability

Vaccine adjuvant MF59 promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells

MF59 is an oil-in-water emulsion adjuvant approved for human influenza vaccination in European Union. The mode of action of MF59 is not fully elucidated yet, but results from several years of investigation indicate that MF59 establishes an immunocompetent environment at injection site which promotes recruitment of immune cells, including antigen presenting cells (APCs), that are facilitated to engulf antigen and transport it to draining lymph node (dLN) where the antigen is accumulated. In vitro studies showed that MF59 promotes the differentiation of monocytes to dendritic cells (Mo-DCs). Since after immunization with MF59, monocytes are rapidly recruited both at the injection site and in dLN and appear to have a morphological change toward a DC-like phenotype, we asked whether MF59 could play a role in inducing differentiation of Mo-DC in vivo. To address this question we immunized mice with the auto-fluorescent protein Phycoerythrin (PE) as model antigen, in presence or absence of MF59. We measured the APC phenotype and their antigen uptake within dLNs, the antigen distribution within the dLN compartments and the humoral response to PE. In addition, using Ovalbumin as model antigen, we measured the capacity of dLN APCs to induce antigen-specific CD4 T cell proliferation. Here, we show, for the first time, that MF59 promotes differentiation of Mo-DCs within dLNs from intranodal recruited monocytes and we suggest that this differentiation could take place in the medullary compartment of the LN. In addition we show that the Mo-DC subset represents the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59. Interestingly, this finding correlates with the enhanced triggering of antigen-specific CD4 T cell response induced by LN APCs. This study therefore demonstrates that MF59 is able to promote an immunocompetent environment also directly within the dLN, offering a novel insight on the mechanism of action of vaccine adjuvants based on emulsions