Dendritic cells (DCs) are major antigen-presenting cells that can efficiently prime and cross-prime antigen-specific T cells. Delivering antigen to DCs via surface receptors is thus an appealing strategy to evoke cellular immunity. Nonetheless, which DC surface receptor to target to yield the optimal CD8+ and CD4+ T cell responses remains elusive. Herein, we report the superiority of CD40 over 9 different lectins and scavenger receptors at evoking antigen-specific CD8+ T cell responses. However, lectins (e.g., LOX-1 and Dectin-1) were more efficient than CD40 at eliciting CD4+ T cell responses. Common and distinct patterns of subcellular and intracellular localization of receptor-bound αCD40, αLOX-1 and αDectin-1 further support their functional specialization at enhancing antigen presentation to either CD8+ or CD4+ T cells. Lastly, we demonstrate that antigen targeting to CD40 can evoke potent antigen-specific CD8+ T cell responses in human CD40 transgenic mice. This study provides fundamental information for the rational design of vaccines against cancers and viral infections.

Influenza virus infection of neonates poses a major health concern, often resulting in severe disease and hospitalization. At present, vaccines for this at-risk population are lacking. Thus, development of an effective vaccine is an urgent need. Here we have used an innovative nonhuman primate neonate challenge model to test the efficacy of a novel TLR 7/8 agonist (R848) conjugated influenza virus vaccine. The use of the intact virus represents a step forward in conjugate vaccine design as it provides multiple antigenic targets allowing for elicitation of a broad immune response. Our results show that this vaccine induces high level virus-specific antibody and cell mediated responses in neonates that result in increased virus clearance and reduced lung pathology following challenge compared to the non-adjuvanted virus vaccine. Surprisingly, the addition of a second TLR agonist (flagellin) did not enhance vaccine protection, suggesting combinations of TLR that provide increased efficacy must be determined empirically. These data support further exploration of this new conjugate influenza vaccine approach as a platform for use in the at-risk neonate population.

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Recurring influenza viruses pose an annual threat to public health. A time-saving, cost-effective and egg-independent influenza vaccine approach is important particularly when responding to an emerging pandemic. We fabricated coated, two-layer protein nanoclusters from recombinant trimeric hemagglutinin from an avian-origin H7N9 influenza A virus as an approach for vaccine development in response to an emerging pandemic. Assessment of the virus-specific immune responses and protective efficacy in mice immunized with the nanoclusters demonstrated that the vaccine candidates were highly immunogenic, able to induce protective immunity and long-lasting humoral antibody responses to this virus without the use of adjuvants. Because the advantages of the highly immunogenic coated nanoclusters also include rapid productions in an egg-independent system, this approach has great potential for influenza vaccine production not only in response to an emerging pandemic, but also as a replacement for conventional seasonal influenza vaccines.

Influenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response following vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for this vulnerable population. To address this issue, we established a neonatal African green monkey (AGM) nonhuman primate model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a Toll-like receptor 5 (TLR5) agonist, to serve as an effective adjuvant in this at-risk population. Four-to 6-day-old AGMs were primed and boosted with inactivated PR8 influenza virus (IPR8) adjuvanted with either wild-type flagellin or inactive flagellin with a mutation at position 229 (m229), the latter of which is incapable of signaling through TLR5. Increased IgG responses were observed following a boost, as well as at early times after challenge, in infants vaccinated with flagellin-adjuvanted IPR8. Inclusion of flagellin during vaccination also resulted in a significantly increased number of influenza virus-specific T cells following challenge compared to the number in infants vaccinated with the m229 adjuvant. Finally, following challenge infants vaccinated with IPR8 plus flagellin exhibited a reduced pathology in the lungs compared to that in infants that received IPR8 plus m229. This study provides the first evidence of flagellin-mediated enhancement of vaccine responses in nonhuman primate neonates. Influenza virus remains one of the leading causes of morbidity and mortality worldwide. Infants less than 6 months of age are particularly vulnerable to development of severe disease following infection (1). Diseases associated with influenza virus infection in children include otitis media, pneumonia, myositis, and croup. While oseltamivir (Tamiflu), one of the two FDA-approved antiinfluenza drugs, can be used in infants aged 2 weeks and older, concerns exist due to the potential for adverse effects, drug resistance, and limited effectiveness in young infants (2).Currently, there are three approved approaches for vaccination against influenza in the United States: intramuscular (i.m.) administration of inactivated influenza virus, intramuscular administration of recombinant hemagglutinin (HA) proteins, and intranasal administration of a live attenuated influenza virus (LAIV). The first is approved for use in individuals aged 6 months and older, the second for use in individuals aged 18 to 49 years, and the last for use in healthy individuals aged 2 to 49 years. Thus, none are approved for use in the vulnerable neonate population. While the lack of approval for the use of these vaccines in the very young may reflect some safety concerns, a principal factor is the poor immune responses elicited in human neonates (3,4).Previous studies, while limited, have shown that an initia...

Our study demonstrated that CS1 plays an inhibitory role in human monocytes to control proinflammatory immune responses.

Influenza infection typically initiates at respiratory mucosal surfaces. Induction of immune responses at the sites where pathogens initiate replication is crucial for the prevention of infection. We studied the adjuvanticity of GPI-anchored CCL28 co-incorporated with influenza HA-antigens in chimeric virus-like particles (cVLPs), in boosting strong protective immune responses through an intranasal (i.n.) route in mice. We compared the immune responses to that from influenza VLPs without CCL28, or physically mixed with soluble CCL28 at systemic and various mucosal compartments. The cVLPs containing GPI-CCL28 showed in-vitro chemotactic activity towards spleen and lung cells expressing CCR3/CCR10 chemokine receptors. The cVLPs induced antigen specific endpoint titers and avidity indices of IgG in sera and IgA in tracheal, lung, and intestinal secretions, significantly higher (4–6 fold) than other formulations. Significantly higher (3–5 fold) hemagglutination inhibition titers and high serum neutralization against H3N2 viruses were also detected with CCL28-containing VLPs compared to other groups. The CCL28-containing VLPs showed complete and 80% protection, when vaccinated animals were challenged with A/Aichi/2/1968/H3N2 (homologous) and A/Philippines/2/1982/H3N2 (heterologous) viruses, respectively. Thus, GPI-anchored CCL28 in influenza VLPs act as a strong immunostimulator at both systemic and mucosal sites, boosting significant cross-protection in animals against heterologous viruses across a large distance.

TX, USA2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM/CD150), is expressed on all NK cells, a subpopulation of T cells, monocytes and basophils. Human NK cells express two isoforms of 2B4, h2B4-A and h2B4-B that differ in a small portion of the extracellular domain. In the present investigation, we have studied the functions of h2B4-A and h2B4-B. Our study demonstrated that these two isoforms differ in their binding affinity for CD48, which results in differential cytotoxic activity as well as intracellular calcium release by NK cells upon target cell recognition. Analysis of the predicted 3-D structure of the two isoforms showed conformational differences that could account for their differences in binding affinity to CD48. h2B4-A was able to mediate natural cytotoxicity against CD48-expressing K562 target cells and induce intracellular calcium release, whereas h2B4-B showed no effects. NK-92MI, U937, THP-1, KU812, primary monocytes, basophils and NK cells showed expression of both h2B4-A and h2B4-B whereas YT and IL-2-activated NK cells did not show any h2B4-B expression. Stimulation of NK cells through 2B4 resulted in decreased mRNA levels of both h2B4-A and h2B4-B indicating that down-regulation of 2B4 isoforms may be an important factor in controlling NK cell activation during immune responses.Key words: Cell activation . Cell surface molecules . NK cells Introduction NK cells are a fundamental component of the innate immune system, capable of recognizing and destroying tumor cells as well as cells that have been infected by viruses or bacteria [1]. In addition to their role in innate immune responses, interactions between NK cells and dendritic cells regulate the adaptive immune response to pathogens [2]. NK cell functions are regulated by a dynamic interplay between activating and inhibitory signals transmitted by distinct classes of receptors found on their surface [3,4]. The balance between positivesignaling receptors and negative-signaling receptors ultimately determines the outcome of NK cell-target cell encounters [5,6].2B4 (CD244), belongs to the signaling lymphocyte-activation molecule (SLAM) family of receptors, which includes SLAM (CD150), CD84, NTB-A (Ly-108), lymphocyte antigen 9 (Ly9; CD229), and CD2-subset 1 (CS1) (also termed CRACC or CD319) [7][8][9][10]. 2B4 is expressed by all NK cells, as well as a subset of memory CD8 1 abT cells, gdT cells, basophils, monocytes and eosinophils [11][12][13][14][15]. 2B4 binds to the glycophosphoinositol anchored protein, CD48, expressed on all hematopoietic cells, with a similar affinity in mouse and human [16,17]. Engagement of 2B4 via interaction with specific antibodies or CD48 induces cytokine secretion (IFN-g) and enhances non-MHC-restricted 1632killing by NK cells [12,16,18]. 2B4 can also function as a ligand on target cells and stimulate NK cells through the engagement of CD48 [19]. 2B4 was initially identified as an activating receptor in non-MHC-restricted killing of NK and T cells [12,20]. Some studies with human ...