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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

Mo-DCs are the major source of antigen-loaded and activated APCs within the dLN when immunizing with MF59.

<p>Popliteal dLN cell suspensions from mice immunized with PE or PE + MF59 analyzed by flow cytometry 18 hrs after immunization. (<b>a</b>) Upper panels: fow cytometry dot plots that show the uptake of PE and MF59 by MΦs (blue cells), DCs (green cells) and monocytes or Mo-DCs (red cells) from one representative experiment in mice immunized with PE or PE + fluorescently labelled-MF59 are reported as example. Middle and lower panels: bar graph histograms report the percentage (middle panels) and the number (per million of live/singlet cells acquired by flow cytometry) (lower panels) of PE positive cells in MΦs (blue bars), DCs (green bars) and monocytes or Mo-DCs (red bars). Data from three independent experiments (single numbered bars) and the arithmetic mean of these values (black horizontal line) are shown. Statistical analysis: parametric one–way ANOVA test (Dunnett’s multiple comparison using Mo-DCs as control column) has been applied to calculate the P-value.<i>*P</i>˂0.05; <i>***P</i>˂0.001; <i>****P</i>˂ 0.0001. (<b>b</b>) Bar graph histogram reports the average (+ standard deviation) number (per million of live/singlet cells acquired by flow cytometry) of PE positive MΦs (blue bars), DCs (green bars) and Mo-DCs vs. monocytes (red bars) in mice immunized with PE (dotted bars) or PE + MF59 (filled bars) in the three independent experiments reported in panel (a). Statistical analysis: parametric one-tailed T-test was used to calculate the P-value. <i>*P</i>˂0.05; **<i>P</i><0.01. (<b>c</b>) Upper panels: flow cytometry histograms of CD80 expression by MΦs (blue), DCs (green) and Mo-DCs vs. monocytes (red) in mice immunized with PE (thin lines) or PE + MF59 (thick lines) from one experiment out of the three shown in panel (a). Lower panel: bar graph histogram shows the difference in CD80 Mean Fluorescence Intensity (MFI) of MΦs (blue bars), DCs (green bars) and Mo-DCs vs. monocytes (red bars) from mice PE immunized with and without MF59 (ΔMFI). Data of the same three independent experiments reported in panel (a) (single numbered bars) and the arithmetic mean of these values (black horizontal line), are shown. Statistical analysis: parametric one-tailed T-test was used to calculate the P-value. <i>**P</i>˂0.01.</p

MF59 promotes differentiation and transient accumulation of Mo-DCs within the dLN.

<p>(<b>a</b>) Left panel: flow cytometry dot plots of dLN cell suspensions to identify APCs are reported for each time point and each treatment (as indicated). Macrophages (MΦ) are depicted in blue, whereas the rest of the APCs are in black. The transient appearance of a CD8α_F4/80 double positive APC subset (red gates) between 8 hrs and 18 hrs after immunization (red quadrant) is shown. Representative results of one experiment out of three are shown. Right panel: bar graph histogram that reports the number x10³ (per million of live/singlet cells acquired by flow cytometry) of CD8α_F4/80 double positive APCs at 8 hrs and 18 hrs after immunization with PE (white bars) and PE + MF59 (grey bars). Data from three independent experiments (single numbered bars) and the arithmetic mean of these values (black horizontal line) are shown. Statistical analysis: parametric one-tailed T-test was used to calculate the P-value. **<i>P</i><0.01. (<b>b</b>) Flow cytometry dot plots of dLN cell suspensions obtained 18 hrs after immunization with PE or PE + MF59 are shown to analyze the expression of the surface protein CD64 on the different APC subsets. CD8α_F4/80 double positive APCs are depicted red, LN DCs in green and MΦs in blue. Representative results of one experiment out of the three independent experiments reported in panel (a) are shown. (<b>c</b>) Flow cytometry dot plots of dLN cell suspensions obtained 8 hrs and 18 hrs after immunization with MF59 reveal that the CD8α_F4/80 double positive APC subset (red cells) expresses the CD11c DC marker 18 hrs but not 8 hrs after the immunization. Representative results of one experiment out of the three reported in panel (a) are shown. (<b>d</b>) Flow cytometry dot plots of the dLN cell suspensions to identify APCs derived from mice immunized with MF59, whose LNs were explanted from both legs 8 hrs after the immunization, but: the LNs from the left legs (left dot plots) were immediately processed (8 hrs), whereas the LNs from the right legs (right dot plots) were processed after 10 hrs of in vitro culture (8 hrs + 10 hrs (in culture)). Representative results of one experiment out of three are shown.</p

Co-localization of antigen, MF59 and DCs within the medullary compartment of the dLN.

<p>(<b>a</b>) Confocal microscopy images of dLNs collected from mice 18 hrs after treatment with PBS, PE or PE + MF59 and labeled to detect DCs and the medullary compartment of the LN. Signals which detect the LN medulla (blue, F4/80), PE (red) and DCs (yellow, CD11c) are shown separately and merged (as indicated). The magnification shows the co-localization of antigen and DCs within the LN medulla. The image of one section is shown, in each panel, as example of consecutive sections of a whole LN, which is representative of the organs of all immunized mice. Original magnification: 5X. Scale bar: 1 mm. Results of one representative experiment out of three are reported. (<b>b</b>) Confocal microscopy images of dLNs collected from mice 18 hrs after treatment with PE + fluorescently labelled-MF59 and labeled to detect DCs and the medullary compartment of the LN. Signals which detect the LN medulla (blue), MF59 (green), PE (red) and DCs (yellow) are shown separately and merged (as indicated). The magnification shows the co-localization of antigen, MF59 and DCs within the LN medulla. The image of one section is shown, in each panel, as example of consecutive sections of a whole LN, which is representative of the organs of all immunized mice. Original magnification: 5X. Scale bar: 1 mm. Representative results of one representative experiment out of two are reported.</p