Distinctive innate immune signatures at injection site boost the adaptive response to flu subunit vaccine.

Vaccine adjuvants activate specific innate immune pathways which boost the adaptive responses to co-administered antigens. Experimental data in mouse and human have shown that the efficacy of vaccine adjuvants is often antigen-dependent. For example, alum has been used for years as an efficient adjuvant for many subunit vaccines including Hepatitis B and Diphteria-Tetanus, but is not very efficient in enhancing the response to flu HA antigen. Recently, several human studies have shown that oil-in-water emulsions such as MF59 are superior to alum in enhancing the response to flu subunit vaccines. However, the mechanism of action of flu adjuvanticity is not fully understood. In this study we wanted to identify the innate immune signatures that are associated to adjuvanticity to flu subunit vaccine in the mouse system. First we immunized mice with seasonal trivalent flu antigens or tetanus toxoid (TT) mixed with MF59, alum or three TLR-dependent adjuvants: the oligonucleotide CpG (TLR9), the small molecule resiquimod (TLR7/8) and the lipopeptide Pam3CSK4 (TLR2). All adjuvants were similarly efficient in inducing an adaptive immune response to TT. By contrast, MF59 and, to a lesser extent, Pam3CSK4, enhanced the antibody responses to HA. In order to correlate innate immune gene signatures induced by the vaccine adjuvants with the ability to boost flu adaptive responses, we performed microarray analysis with the same set of adjuvants both in vitro in mouse splenocytes and in vivo in mouse muscle and draining lymph nodes. Unlike TLR agonists, MF59 and alum did not affect gene expression of blood cells in vitro and draining LN in vivo. On the other hand, MF59 was the strongest activator of cytokine, chemokine and leukocyte transendothelial migration genes in the muscle, suggesting an early and stronger cell recruitment potential. Indeed MF59 and Pam3CSK induced a stronger and more rapid recruitment of CD11b+ cells (monocytes, DCs and granulocytes) at injection site compared to the other adjuvants tested. It has been recently proposed that TLR7-dependent activation of the interferon type 1 pathway is the innate immune pathway required for adjuvanticity to flu vaccines. Surprisingly, we found that the TLR7 adjuvant resiquimod did not affect the adaptive response to flu despite a strong activation of the interferon pathway in the muscle and in the LNs. In summary, here we show that strong activation of innate immunity in the muscle, but not in draining LNs, and local recruitment of CD11b+ blood cells at injection site by MF59 and TLR2 vaccine adjuvants correlates with their ability to enhance antibody responses to flu antigens.

Studio in modello animale della regolazione trascrizionale indotta dalla somministrazione di alcune molecole con attività immunostimolatoria. In particolare il lavoro è focalizzato sull’effetto che ha l’emulsione MF59 sull’immunità innata a livello del sito di iniezione, rispetto ad altre classi di adiuvanti. Dati riguardanti la risposta anticorpale hanno dimostrato con MF59 sia il miglior adiuvante per gli antigeni dell’influenza rispetto agli altri adiuvanti classici come alum e CpG. Per questo si è deciso di investigare, tramite analisi microarray, l’effetto di MF59, rispetto alle altre molecole, a livello di espressione genica al sito di iniezione (nel muscolo) e distalmente (nel linfonodo). È stato osservato come MF59 sia il più forte attivatore di geni codificanti per chemochine e citochine al sito di iniezione, suggerendo un suo effetto sull’immunità innata. Essendo inoltre l’emulsione anche in grado di attivare l’espressione di geni codificanti per proteine coivolte nel pathway di migrazione transendoteliale e per i recettori delle chemochine, è stato selezionato il gene Itgam, codificante per l’integrina CD11b, appunto coinvolta in tale meccanismo: lo studio di localizzazione tramite microscopia confocale di sezioni di muscolo, ha evidenziato come MF59, rispetto agli altri adiuvanti, induca un “recruitment” di cellule CD11b+ al sito di iniezione. Inoltre la regolazione dell’espressione di alcuni marcatori di superficie in cellule del linfonodo è stata confermata mediante citofluorimetria.