Buying Time—The Immune System Determinants of the Incubation Period to Respiratory Viruses

Respiratory viruses cause disease in humans characterized by an abrupt onset of symptoms. Studies in humans and animal models have shown that symptoms are not immediate and appear days or even weeks after infection. Since the initial symptoms are a manifestation of virus recognition by elements of the innate immune response, early virus replication must go largely undetected. The interval between infection and the emergence of symptoms is called the incubation period and is widely used as a clinical score. While incubation periods have been described for many virus infections the underlying mechanism for this asymptomatic phase has not been comprehensively documented. Here we review studies of the interaction between human pathogenic respiratory RNA viruses and the host with a particular emphasis on the mechanisms used by viruses to inhibit immunity. We discuss the concept of the “stealth phase”, defined as the time between infection and the earliest detectable inflammatory response. We propose that the “stealth phase” phenomenon is primarily responsible for the suppression of symptoms during the incubation period and results from viral antagonism that inhibits major pathways of the innate immune system allowing an extended time of unhindered virus replication

Intensified and protective CD4+ T cell immunity in mice with anti–dendritic cell HIV gag fusion antibody vaccine

Current human immunodeficiency virus (HIV) vaccine approaches emphasize prime boost strategies comprising multiple doses of DNA vaccine and recombinant viral vectors. We are developing a protein-based approach that directly harnesses principles for generating T cell immunity. Vaccine is delivered to maturing dendritic cells in lymphoid tissue by engineering protein antigen into an antibody to DEC-205, a receptor for antigen presentation. Here we characterize the CD4+ T cell immune response to HIV gag and compare efficacy with other vaccine strategies in a single dose. DEC-205–targeted HIV gag p24 or p41 induces stronger CD4+ T cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of interferon (IFN)-γ– and interleukin 2–producing CD4+ T cells are elicited, including double cytokine-producing cells. In addition, the response is broad because the primed mice respond to an array of peptides in different major histocompatibility complex haplotypes. Long-lived T cell memory is observed. After subcutaneous vaccination, CD4+ and IFN-γ–dependent protection develops to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. We suggest that a DEC-targeted vaccine, in part because of an unusually strong and protective CD4+ T cell response, will improve vaccine efficacy as a stand-alone approach or with other modalities

Unique Type I Interferon Responses Determine the Functional Fate of Migratory Lung Dendritic Cells during Influenza Virus Infection

Migratory lung dendritic cells (DCs) transport viral antigen from the lungs to the draining mediastinal lymph nodes (MLNs) during influenza virus infection to initiate the adaptive immune response. Two major migratory DC subsets, CD103+ DCs and CD11bhigh DCs participate in this function and it is not clear if these antigen presenting cell (APC) populations become directly infected and if so whether their activity is influenced by the infection. In these experiments we show that both subpopulations can become infected and migrate to the draining MLN but a difference in their response to type I interferon (I-IFN) signaling dictates the capacity of the virus to replicate. CD103+ DCs allow the virus to replicate to significantly higher levels than do the CD11bhigh DCs, and they release infectious virus in the MLNs and when cultured ex-vivo. Virus replication in CD11bhigh DCs is inhibited by I-IFNs, since ablation of the I-IFN receptor (IFNAR) signaling permits virus to replicate vigorously and productively in this subset. Interestingly, CD103+ DCs are less sensitive to I-IFNs upregulating interferon-induced genes to a lesser extent than CD11bhigh DCs. The attenuated IFNAR signaling by CD103+ DCs correlates with their described superior antigen presentation capacity for naïve CD8+ T cells when compared to CD11bhigh DCs. Indeed ablation of IFNAR signaling equalizes the competency of the antigen presenting function for the two subpopulations. Thus, antigen presentation by lung DCs is proportional to virus replication and this is tightly constrained by I-IFN. The “interferon-resistant” CD103+ DCs may have evolved to ensure the presentation of viral antigens to T cells in I-IFN rich environments. Conversely, this trait may be exploitable by viral pathogens as a mechanism for systemic dissemination

Regulatory T cell ablation causes acute T cell lymphopenia.

Regulatory T (Treg) cells enforce T cell homeostasis and maintain peripheral T cell tolerance. Here we report a previously unappreciated phenomenon of acute T cell lymphopenia in secondary lymphoid organs and non-lymphoid tissues triggered by Treg cell depletion that precedes the expansion of self-reactive T cells. Lymphopenia affects both neonates and adults indicating a dominant role of Treg cells in maintaining peripheral T cell numbers regardless of the developmental stage. The lymphopenia was neither triggered by caspase-dependent apoptosis nor macrophage-mediated clearance of T cells, nor diminished survival of naïve or recently activated T cells due to paucity of IL-7. It is possible that transient lymphopenia associated with congenital or acute Treg cell deficiency may contribute to the development of T cell mediated autoimmune disorders

Transient acute T cell lymphopenia induced by Treg cell ablation in adult mice.

<p>Adult <i>Foxp3^DTR</i> mice were injected i.p. with DT or PBS (control) and spleen, lymph nodes, blood, lung and livers were collected 2 days later. (<b>A</b>) Absolute numbers of TCRβ⁺ T cells (left column), (<b>B</b>) CD4⁺ T cells (middle column), and (<b>C</b>) CD8⁺ T cells (right column) in the blood, spleen, lymph nodes, lung and liver of DT- (closed squares) or control PBS-treated (open squares) <i>Foxp3^DTR</i> mice. Results are representative of at least 2 independent experiments (n = 5 mice per group).</p

Apoptotic T cell death or MF engulfment do not account for Treg cell depletion mediated transient lymphopenia.

<p>Spleen and lymph node cells from DT- or PBS-treated <i>Foxp3^DTR</i> mice were stained with Annexin-V and analyzed by flow cytometry. Percentage of Annexin-V⁺ CD4⁺ (<b>A</b>) and CD8⁺ T cells (<b>B</b>) in DT- (closed circles) or PBS-treated (open circles) <i>Foxp3^DTR</i> mice. (<b>C</b>) Caspase inhibitor QVD-OPH was administered i.p. into <i>Foxp3^DTR</i> mice on the day of, and on day 1 after DT injection. Percentages of splenic TCRβ⁺ T cells in QVD-OPH treated or control mice on day 2 after DT or PBS treatment. Clodronate encapsulated (Clo-lip) or control (PBS-lip) liposomes were injected i.v. into <i>Foxp3^DTR</i> mice 10 days prior to DTx. 2 days after DT injection, splenic T cells were analyzed by flow cytometry. (<b>D</b>) Flow cytometric analysis of red pulp MF depletion in Clo-lip treated mice. Percentage of red pulp MF depletion (<b>E</b>) and spleen TCRβ⁺ T cells (<b>F</b>) in DT treated <i>Foxp3^DTR</i> mice injected with Clo-lip (closed red squares) or PBS-lip (closed black squares) and in PBS treated <i>Foxp3^DTR</i> mice injected with Clo-lip (open red squares) or PBS-lip (open black squares). Results are representative of at least 2 independent experiments (n≥3 per group).</p

Treg cell ablation has no apparent impact on thymic function in neonates.

<p>Neonates were injected i.p with DT or PBS on day 3 after birth, and cell suspensions from the thymus were prepared on day 7 after birth. <b>A.</b> FACS analysis of CD4⁺ and CD8⁺ single positive (SP), double positive (DP) and double negative (DN) thymocytes. <b>B.</b> Comparison of thymocyte absolute numbers in DT-treated and PBS control groups. <b>C–E.</b> Absolute numbers of DNs, DPs and CD8⁺ SPs, respectively. <b>F–H.</b> Absolute numbers of CD4⁺ TCRβ⁺ SP, immature CD4⁺ CD24^hi CD62L^lo and mature CD4⁺ CD24^lo CD62L^hi SPs. <b>I.</b> Flow cytometry analysis of CD4⁺ SP subsets, as indicated in (G and H). <b>J.</b> Thymic T cell output in neonates was measured by the direct injection to the thymus of 20 µL of Alexa Fluor-647 succinimidyl ester at 10 mg/mL followed by an i.p injection of DT or PBS. Scatter plot representing the ratios of Alexa-647⁺ CD4⁺ T cells in the thymus and spleen at day 7 after birth. Results are representative of at least 2 independent experiments (n≥3 mice per group).</p

miR-128 Is Implicated in stress Response by Targeting MAFG in Skeletal Muscle Cells

MAFG (v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog G) is a bZIP-type transcriptional regulator that belongs to the small MAF (sMAFs) protein family. By interacting with other bZIP transcription factors, sMAFs can form homo- and heterodimers governing either repressive or activating transcriptional functions. As heterodimeric partner of Nrf2, MAFG positively influences the ARE-dependent antioxidant/xenobiotic pathways, at least in condition of a correct MAFG:Nrf2 balance. MicroRNAs (miRs) participate to different regulatory networks being involved as fine-tuning regulators of gene expression. However, the connections between cellular surveillance to stresses mediated by MAFG:Nrf2 and miR regulations are not well understood. Here, we explored the impact of miR-128 in expression of genes related to stress response. Bioinformatic predictions coupled with functional analysis revealed the presence of miR-128 binding site in the 3′UTR of MAFG. Ectopic miR-128 expression correlated with reduced expression of endogenous MAFG-dependent genes and negatively affected ARE-mediated molecular phenotype based on Nrf2 activity. Indeed, miR-128 impairs redox-dependent pathways induced in response to oxidative stress. Moreover, in condition of hypoxia, MAFG induction correlated with reduced levels of miR-128. This lead to increased mRNA levels of HMOX-1 and x-CT for blunting stress. Overall, these findings identify MAFG as novel direct target of miR-128

Optimization of Human Immunodeficiency Virus Gag Expression by Newcastle Disease Virus Vectors for the Induction of Potent Immune Responses ▿

One attractive strategy for the development of a human immunodeficiency virus (HIV) vaccine is the use of viral vectors with a proven safety profile and an absence of preexisting immunity in humans, such as Newcastle disease virus (NDV). Several NDV vaccine vectors have been generated, and their immunogenicities have been investigated with different animal models. However, a systematic study to evaluate the optimal insertion site of the foreign antigens into NDV that results in enhanced immune responses specific to the antigen has not yet been conducted. In this article, we describe the ability of NDV expressing HIV Gag to generate a Gag-specific immune response in mice. We also have determined the optimal insertion site into the NDV genome by generating recombinant NDV-HIVGag viruses in which HIV gag was located at different transcriptional positions throughout the NDV viral genome. All recombinant viruses were viable, grew to similar titers in embryonated chicken eggs, and expressed Gag in a stable manner. Our in vivo experiments revealed that higher HIV Gag protein expression positively correlates with an enhanced CD8+ T-cell-mediated immune response and protective immunity against challenge with vaccinia virus expressing HIV Gag. We also inserted a codon-optimized version of HIV gag in the described best location, between the P and M genes. Virus expressing the codon-optimized version of HIV gag induced a higher expression of the protein and an enhanced immune response against HIV Gag in mice. These results indicate that strategies directed toward increasing antigen expression by NDV result in enhanced immunogenicity and vaccine efficacy

Hemocyanins as immunostimulants Hemocianinas, una herramienta inmunológica de la biomedicina actual

Hemocyanins, the giant oxygen transporter glycoproteins of diverse mollusks, are xenogenic to the mammalian immune system and they display a remarkable immunogenicity. Therefore they are ideal non-specific immunostimulants to treat some types of cancer. They are used as an alternative therapy for superficial urinary bladder cancer (SBC), that has been traditionally treated with Bacillus Calmette-Guèrin (BCG). In contrast to BCG, hemocyanins do not cause side-effects, making them ideal for long-term repetitive treatments. Hemocyanins have also been exploited as carriers to develop antibodies against hapten molecules and peptides, as carrier-adjuvants for cutting-edge vaccines against cancer, drug addiction, and infectious diseases and in the diagnosis of parasitic diseases, such as Schistosomiasis. The hemocyanin from Megathura crenulata, also known as keyhole limpet hemocyanin (KLH), has been used for over thirty years for the purposes described above. More recently, hemocyanin from t