Targeting M2e to DEC-205 induces an enhanced serum antibody-dependent heterosubtypic protection against influenza A virus infection.

The ectodomain of the influenza A virus (IAV) M2 protein (M2e) is highly conserved, and it represents a promising candidate for the development of an "universal vaccine". However, the low immunogenicity associated to M2e in a natural infection or in response to seasonal vaccines has led to explore new approaches to enhance it. In recent years, it has become clear that targeting antigens to dendritic cells (DC) is an efficient way to enhance immune responses against pathogens. In this work, the M2e peptide was chemically cross-linked to a monoclonal antibody (mAb) specific for DEC-205 (α-DEC-205:M2e), present on DC. BALB/c mice were inoculated subcutaneously (s.c.) three times with the conjugate equivalent to 1 µg of M2e, in the presence of polyinosinic-polycytidylic acid (poly I:C) as adjuvant. As controls, other groups of mice were inoculated under the same conditions with M2e cross-linked to an isotype control mAb (isotype:M2e), 5 µg of free M2e peptide, ovalbumin (OVA) cross-linked to the α-DEC-205 mAb (α-DEC-205:OVA) or poly I:C alone. Immunization with α-DEC-205-M2e induced high levels of serum antibodies (Abs) compared to isotype:M2e or to free M2e peptide, and in all cases IgG1 was predominant over IgG2a Abs. Furthermore, immunization with the α-DEC-205:M2e conjugate did not prevent morbidity, but it induced up to 76% protection against a heterosubtypic IAV lethal challenge. Contrasting with the 20 to 40% protection induced by isotype:M2e or by free M2e peptide. The protection induced by α-DEC-205:M2e conjugate was dependent on non-neutralizing serum Abs and independent of effector CD4+ T cells. These results show that targeting M2e to DEC-205 is a very effective alternative to induce strong heterosubtypic protection against an IAV infection.

Enhancement of VP6 immunogenicity and protective efficacy against rotavirus by VP2 in a genetic immunization.

VP2/VP6 virus like particles (VLPs) are very effective in inducing protection against the rotavirus infection in animal models. Individually, VP6 can also induce protection. However, there is no information about the immunogenicity of VP2. The aim of this work was to evaluate the efficacy of DNA vaccines codifying for VP2 or VP6, alone or combined, to induce protection against the rotavirus infection. Murine rotavirus VP2 and VP6 genes were cloned into the pcDNA3 vector. Adult BALB/c mice were inoculated three times by intramuscular (i.m.) injections with 100 or 200µg of pcDNA3-VP2 or pcDNA3-VP6 alone or co-administered with 100µg of pcDNA3-VP2/100µg of pcDNA3-VP6. Two weeks after the last inoculation, mice were challenged with the wild type murine rotavirus strain epizootic diarrhea of infant mice (EDIMwt). We found that both plasmids, pcDNA3-VP2 and pcDNA3-VP6, were able to induce rotavirus-specific serum antibodies, but not intestinal rotavirus-specific IgA; only 200µg of pcDNA3-VP6 induced 35% protection against the infection. A similar level of protection was found when mice were co-administered with 100µg of pcDNA3-VP2/100µg of pcDNA3-VP6 (1:1 ratio). However, the best protection (up to 58%) occurred when mice were inoculated with 10µg of pcDNA3-VP2/100µg of pcDNA3-VP6 (1:10 ratio). These results indicate that the DNA plasmid expressing VP6 is a better vaccine candidate that the one expressing VP2. However, when co-expressed, VP2 potentiates the immunogenicity and protective efficacy of VP6.

Rotavirus activates dendritic cells derived from umbilical cord blood monocytes.

Rotavirus is the most common cause of acute infectious diarrhea in human neonates and infants. However, the studies aimed at dissecting the anti-virus immune response have been mainly performed in adults. Dendritic cells (DCs) play a crucial role in innate and acquired immune responses. Therefore, it is very important to determine the response of neonatal and infant DCs to rotavirus and to compare it to the response of adult DCs. Thus, we determined the response of monocyte-derived DCs from umbilical cord blood (UCB) and adult peripheral blood (PB) to rotavirus in vitro. It was found that the rotavirus and its genome, composed of segmented doubled stranded RNA (dsRNA), induced the activation of neonatal DCs, as these cells up-regulated the levels of CD40, CD86, MHC II, TLR-3 and TLR-4, the production of cytokines IL-6, IL-12/23p40, IL-10, TGF-ß (but not of IL-12p70), and the message for TNF-α and IFN-ß. This activation enabled the neonatal DCs to induce a strong proliferation of allogeneic CD4+ T cells and the production of IFN-γ. Moreover, neonatal DCs could be infected by rotavirus and sustain its replication. Neonatal DCs had a similar response as adult DCs towards rotavirus and its genome. However, adult DCs had a biased pro-inflammatory response compared to neonatal DCs, which showed a biased regulatory profile, as they produced higher levels of IL-10 and TGF-ß, and were less efficient in inducing a Th1 type response. So it can be concluded that rotavirus and its genome can induce the activation of neonatal DCs in spite of their tolerogenic bias.

Targeting of rotavirus VP6 to DEC-205 induces protection against the infection in mice.

Rotavirus (RV) is the primary etiologic agent of severe gastroenteritis in human infants. Although two attenuated RV-based vaccines have been licensed to be applied worldwide, they are not so effective in low-income countries, and the induced protection mechanisms have not been clearly established. Thus, it is important to develop new generation vaccines that induce long lasting heterotypic immunity. VP6 constitutes the middle layer protein of the RV virion. It is the most conserved protein and it is the target of protective T-cells; therefore, it is a potential candidate antigen for a new generation vaccine against the RV infection. We determined whether targeting the DEC-205 present in dendritic cells (DCs) with RV VP6 could induce protection at the intestinal level. VP6 was cross-linked to a monoclonal antibody (mAb) against murine DEC-205 (αDEC-205:VP6), and BALB/c mice were inoculated subcutaneously (s.c.) twice with the conjugated containing 1.5 µg of VP6 in the presence of polyinosinic-polycytidylic acid (Poly I:C) as adjuvant. As controls and following the same protocol, mice were immunized with ovalbumin (OVA) cross-linked to the mAb anti-DEC-205 (αDEC-205:OVA), VP6 cross-linked to a control isotype mAb (Isotype:VP6), 3 µg of VP6 alone, Poly I:C or PBS. Two weeks after the last inoculation, mice were orally challenged with a murine RV. Mice immunized with α-DEC-205:VP6 and VP6 alone presented similar levels of serum Abs to VP6 previous to the virus challenge. However, after the virus challenge, only α-DEC-205:VP6 induced up to a 45% IgA-independent protection. Memory T-helper (Th) cells from the spleen and the mesenteric lymph node (MLN) showed a Th1-type response upon antigen stimulation in vitro. These results show that when VP6 is administered parenterally targeting DEC-205, it can induce protection at the intestinal level at a very low dose, and this protection may be Th1-type cell dependent.