CD11c⁺ cells primed with unrelated antigens facilitate an accelerated immune response to influenza virus in mice.

Recent evidence suggests that an individual's unique history and sequence of exposures to pathogens and antigens may dictate downstream immune responses to disparate antigens. We show that the i.n. delivery of nonreplicative virus-like particles (VLPs), which bear structural but no antigenic similarities to respiratory pathogens, acts to prime the lungs of both C56BL/6 and BALB/c mice, facilitating heightened and accelerated primary immune responses to high-dose influenza challenge, thus providing a nonpathogenic model of innate imprinting. These responses correspond closely to those observed following natural infection with the opportunistic fungus, Pneumocystis murina, and are characterized by accelerated antigen processing by DCs and alveolar macrophages, an enhanced influx of cells to the local tracheobronchial lymph node, and early upregulation of T-cell co-stimulatory/adhesion molecules. CD11c⁺ cells, which have been directly exposed to VLPs or Pneumocystis are necessary in facilitating enhanced clearance of influenza virus, and the repopulation of the lung by Ly-6C⁺ precursors relies on CCR2 expression. Thus, immune imprinting 72 h after VLP-priming, or 2 weeks after Pneumocystis-priming is CCR2-mediated and results from the enhanced antigen processing, maturation, and trafficking abilities of DCs and alveolar macrophages, which cause accelerated influenza-specific primary immune responses and result in superior viral clearance.

Melanoma and lymphocyte cell-specific targeting incorporated into a heat shock protein cage architecture.

Protein cages, including viral capsids, ferritins, and heat shock proteins (Hsps), can serve as nanocontainers for biomedical applications. They are genetically and chemically malleable platforms, with potential as therapeutic and imaging agent delivery systems. Here, both genetic and chemical strategies were used to impart cell-specific targeting to the Hsp cage from Methanococcus jannaschii. A tumor vasculature targeting peptide was incorporated onto the exterior surface of the Hsp cage. This protein cage bound to alpha(v)beta(3) integrin-expressing cells. Cellular tropism was also imparted by conjugating anti-CD4 antibodies to the exterior of Hsp cages. These Ab-Hsp cage conjugates specifically bound to CD4(+) cells. Protein cages have the potential to simultaneously incorporate multiple functionalities, including cell-specific targeting, imaging, and therapeutic agent delivery. We demonstrate the simultaneous incorporation of two functionalities, imaging and cell-specific targeting, onto the Hsp protein cage.

Biomimetic antigenic nanoparticles elicit controlled protective immune response to influenza.

Here we present a biomimetic strategy toward nanoparticle design for controlled immune response through encapsulation of conserved internal influenza proteins on the interior of virus-like particles (VLPs) to direct CD8+ cytotoxic T cell protection. Programmed encapsulation and sequestration of the conserved nucleoprotein (NP) from influenza on the interior of a VLP, derived from the bacteriophage P22, results in a vaccine that provides multistrain protection against 100 times lethal doses of influenza in an NP specific CD8+ T cell-dependent manner. VLP assembly and encapsulation of the immunogenic NP cargo protein is the result of a genetically programmed self-assembly making this strategy amendable to the quick production of vaccines to rapidly emerging pathogens. Addition of adjuvants or targeting molecules were not required for eliciting the protective response.

Inducible bronchus-associated lymphoid tissue (iBALT) synergizes with local lymph nodes during antiviral CD4+ T cell responses.

BACKGROUND: Exposure of the lungs to an antigen or pathogen elicits the formation of lymphoid satellite islands termed inducible bronchus-associated lymphoid tissue (iBALT). However, little is known about how the presence of iBALT, induced by a stimulus unrelated to the subsequent challenge agent, influences systemic immunity in distal locations, whether it be independently, antagonistically, or synergistically. Here, we determined the kinetics of the influenza-specific responses in the iBALT, tracheobronchial lymph node (TBLN), and spleen of mice with and without pre-formed iBALT. METHODS AND RESULTS: Mice with VLP-induced iBALT or no pre-formed iBALT were challenged with influenza. We found that, as we have previously described, those mice whose lungs contained pre-formed iBALT were protected from morbidity, and furthermore, that these mice had increased dendritic cell, and alveolar macrophage accumulation in both the iBALT and TBLNs. This translated to similarly accelerated kinetics and intensified influenza-specific CD4(+), but not CD8(+) T cell responses in the iBALT, TBLN, and spleen. This expansion was then followed by a more rapid T cell contraction in all lymphoid tissues in the mice with pre-formed iBALT. CONCLUSIONS: Thus, iBALT itself may not be responsible for the accelerated primary immune response we observe in mice with pre-formed iBALT, but may contribute to an overall accelerated local and systemic primary CD4(+), but not CD8(+) T cell response. Furthermore, less damaging immune responses observed in mice with pre-formed iBALT may be due to a quicker contraction of CD4(+) T cell responses in both local and systemic secondary lymphoid tissue.

A virus-like particle vaccine platform elicits heightened and hastened local lung mucosal antibody production after a single dose.

We show that a model antigen, ovalbumin (OVA), can be chemically conjugated to the exterior of a small heat shock protein (sHsp) cage that has structural similarities to virus-like particles (VLPs). OVA-sHsp conjugation efficiency was dependent upon the stoichiometry and the length of the small molecule linker utilized, and the attachment position on the sHsp cage. When conjugated OVA-sHsp was delivered intranasally to naïve mice, the resulting immune response to OVA was accelerated and intensified, and OVA-specific IgG1 responses were apparent within 5 days after a single immunizing dose, illustrating its utility for vaccine development. If animals were pretreated with a disparate VLP, P22 (a non-replicative bacteriophage capsid), before OVA-sHsp conjugate immunization, OVA-specific IgG1 responses were apparent already by 4 days after a single immunizing dose of conjugate in OVA-naïve mice. Additionally, the mice pretreated with P22 produced high titer mucosal IgA, and isotype-switched OVA-specific serum IgG. Similarly, sHsp pretreatment enhanced the accumulation of lung germinal center B cells, T follicular helper cells, and increased polymeric Ig receptor expression, priming the lungs for subsequent IgG and IgA responses to influenza virus challenge. Thus, sHsp nanoparticles elicited quick and intense antibody responses and these accelerated responses could similarly be induced to antigen chemically conjugated to the sHsp. Pretreatment of mice with P22 further accelerated the onset of the antibody response to OVA-sHsp, demonstrating the utility of conjugating antigens to VLPs for pre-, or possibly post-exposure prophylaxis of lung, all without the need for adjuvant.

Type I interferon signaling and B cells maintain hemopoiesis during Pneumocystis infection of the lung.

Loss of CD4 T cells is the hallmark of HIV infection. However, type I IFN-producing plasmacytoid dendritic cells may also be lost. This results in susceptibility to an opportunistic infection such as Pneumocystis pneumonia. In addition, regenerative bone marrow failure resulting in pancytopenia is another common problem in advanced stage AIDS. This may be linked to both the failing immune system and recurrent opportunistic infections. We generated lymphocyte-deficient type I IFN receptor-deficient mice (IFrag-/-) to study the effects on Pneumocystis infection of the lung. When IFrag-/- animals were infected with Pneumocystis they died between days 16 and 21 postinfection with minimal pneumonia but severe anemia due to complete bone marrow failure. This included the loss of uncommitted hemopoietic precursor cells. Bone marrow failure was prevented by the reconstitution of IFrag-/- mice with wild-type lymphocytes, especially B cells. T and B cells lacking type I IFN receptor signaling could only partially prevent bone marrow failure in response to Pneumocystis infection. However, the presence of T and B cells lacking type I IFN signaling resulted in compensatory extramedullary hemopoiesis in the liver and spleen. Lymphocyte support of the regenerative capacity of the bone marrow was provided by both type I IFN-dependent and -independent mechanisms that acted synergistically. Our findings point to the requirement of both type I IFNs and lymphocytes in the regenerative capabilities of the hemopoietic system under the pressure of Pneumocystis infection, but not during steady-state hemopoiesis. This may have implications in the management of pancytopenia in AIDS.

Biodistribution studies of protein cage nanoparticles demonstrate broad tissue distribution and rapid clearance in vivo.

Protein cage nanoparticles have the potential to serve as multifunctional cell targeted, imaging and therapeutic platforms for broad applications in medicine. However, before they find applications in medicine, their biocompatibility in vivo needs to be demonstrated. We provide here baseline biodistribution information of two different spherical protein cage nanoplatforms, the 28 nm viral Cowpea chlorotic mottle virus (CCMV) and the 12 nm heat shock protein (Hsp) cage. In naive and immunized mice both nanoplatforms show similar broad distribution and movement throughout most tissues and organs, rapid excretion, the absence of long-term persistence within mice tissue and organs, and no overt toxicity after a single injection. These results suggest that protein cage based nanoparticles may serve as safe, biocompatible, nanoplatforms for applications in medicine.