Adverse effect of vaccination in xenogeneic animals.

Lumpy skin disease (LSD) is a devastating, emerging viral disease of cattle. It causes significant economic losses due to trade restrictions that are placed on infected animals and the biological effects of the disease: infertility, dramatic loss in milk production, induction of abortion and mortality. It is caused by lumpy skin disease virus (LSDV), which belongs to the Poxviridae family. Vaccination has been determined to be the most effective way to control LSD infection among livestock. However, some adverse effects have been reported in animals vaccinated with live vaccines. To the best of our knowledge, this is the first study to report the systemic lesions that are associated with LSD vaccination in xenogeneic animals. The aim of our study was to compare the immunogenicity and pathogenicity of a live attenuated vaccine of Romanian strain of sheeppox virus (SPPV) through study of two different routes of administration in xenogeneic animals (mice). Swiss male mice were inoculated with two doses of SPPV vaccine by two different routes intranasal (IN, through nebulisation), and intraperitoneal (IP) injection) and the levels of immunoglobulins and histopathological findings were reported. Our results showed marked increases in levels of immunoglobulins (Ig) dependent on the administration route: IgG in IP-inoculated mice and IgA in IN-vaccinated mice. IgM levels became markedly high after vaccination via both routes. Histologically, nebulisation of mice with SPPV vaccine caused more pulmonary lesions than did IP injection and promoted the proliferation of megakaryocytes in splenic tissues. In contrast, IP injection had less effect on pulmonary tissues and induced activation of extramedullary haematopoiesis (EH) in the hepatic tissues. LSD vaccination in xenogeneic animals caused serious systemic complications and the severity of the lesions caused to tissue depended on the route of administration.

Combination rhIL-15 and Anti-PD-L1 (Avelumab) Enhances HIVGag-Specific CD8 T-Cell Function.

In chronic HIV infection, virus-specific cytotoxic CD8 T cells showed expression of checkpoint receptors and impaired function. Therefore, restoration of CD8 T-cell function is critical in cure strategies. Here, we show that in vitro blockade of programmed cell death ligand 1 (PD-L1) by an anti-PD-L1 antibody (avelumab) in combination with recombinant human interleukin-15 (rhIL-15) synergistically enhanced cytokine secretion by proliferating HIVGag-specific CD8 T cells. In addition, these CD8 T cells have a CXCR3+PD1-/low phenotype, suggesting a potential to traffic into peripheral tissues. In vitro, proliferating CD8 T cells express PD-L1 suggesting that anti-PD-L1 treatment also targets virus-specific CD8 T cells. Together, these data indicate that rhIL-15/avelumab combination therapy could be a useful strategy to enhance CD8 T-cell function in cure strategies.

Recombinant Human Interleukin-15 and Anti-PD-L1 Combination Therapy Expands a CXCR3+PD1-/low CD8 T-Cell Subset in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

BACKGROUND: The PD1/PD-L1 pathway contributes to the pathogenesis of human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection, and blockade of this pathway may have potential to restore immune function and promote viral control or elimination. In this study, we combined a checkpoint inhibitor anti-PD-L1 (Avelumab) and recombinant human interleukin-15 (rhIL-15) in SIV-infected rhesus macaques (RM). METHODS: The rhIL-15 was administered as continuous infusion in 2 cycles of 10 days in the context of weekly administration of anti-PD-L1 (Avelumab) in SIV-infected RM receiving combination antiretroviral therapy (cART). Safety, immunological parameters, and viral loads were monitored during the study. RESULTS: Administration of rhIL-15/anti-PD-L1 was safe and well tolerated. Treatment resulted in transient increases in proliferating (Ki67+) natural killer and CD8 T cells. In addition, treatment expanded a CXCR3+PD1-/low CD8 T-cell subset with the ability to secrete cytokines. Despite these effects, no changes in plasma viremia were observed after cART interruption. CONCLUSIONS: Expansion of the CXCR3+PD1-/low CD8 T-cell subset with functional capacity and potential to traffic to sites of viral reservoirs in SIV-infected rhesus macaques had no demonstrable effect on plasma viremia after cART interruption.

Insight on thermal stress response of demosponge Chondrosia reniformis (Nardo, 1847).

Global warming has led to an increase in extreme weather and climate phenomena, including floods and heatwaves. Marine heatwaves have frightening consequences for coastal benthic communities around the world. Each species exhibits a natural range of thermal tolerance and responds to temperature variations through behavioral, physiological, biochemical, and molecular adjustments. Physiological stress leading to disease and mass mortality appears when tolerance thresholds are exceeded. Sessile species are therefore particularly affected by these phenomena. Among these sessile species, marine sponges are important members of coral reef ecosystems. To better understand the sponge thermal stress response, we tested the response of demosponge Chondrosia reniformis (Nardo, 1847) to three different temperatures (8 °C, 24 °C and 30 °C) during two exposure periods of time (4 and 14 h). Histological studies of whole parts of the sponge, biochemical analyses (Defense enzymes) and gene expression levels of some target genes were undertaken in this study. The exposure to cold temperature (8 °C) resulted in inhibition of antioxidant enzymes and less modification in the gene expression level of the heat shock proteins (HSPs). These latter were strongly upregulated after exposure to a temperature of 24 °C for 4 h. However, exposure to 30 °C at both periods of time resulted in indication of HSP, antioxidant enzymes, the gene involved in the apoptosis process (Bcl-2: B-cell lymphoma 2), the gene involved in inflammation (TNF: Tumor Necrosis Factor), as well as the aquaporin gene, involved in H2O2 permeation. Moreover, the normal organization of the whole organism was disrupted by the extension and fusion of choanocyte chambers and alteration of the pinacoderm. Interestingly, exposure to sublethal temperatures may show that this sponge has an adaptation threshold temperature. These insights into the adaptation mechanisms of sponges contribute to better management and conservation of sponges and to the prediction of ecosystem trajectories with future climate change.

First insight of genetic diversity, phylogeographic relationships, and population structure of marine sponge Chondrosia reniformis from the eastern and western Mediterranean coasts of Tunisia.

Despite the strategic localization of Tunisia in the Mediterranean Sea, no phylogeographic study on sponges has been investigated along its shores. The demosponge Chondrosia reniformis, descript only morphologically along Tunisian coasts, was chosen to estimate the influence of natural oceanographic and biogeographic barriers on its genetic differentiation and its Phylogeography. The cytochrome oxidase subunit I (COI) gene was amplified and analyzed for 70 Mediterranean Chondrosia reniformis, collected from eight localities in Tunisia. Polymorphism results revealed high values of haplotype diversity (H d) and very low nucleotide diversity (π). Thus, these results suggest that our sponge populations of C. reniformis may have undergone a bottleneck followed by rapid demographic expansion. This suggestion is strongly confirmed by the results of neutrality tests and "mismatch distribution." The important number of haplotypes between localities and the high genetic differentiation (F st ranged from 0.590 to 0.788) of the current C. reniformis populations could be maintained by the limited gene flow Nm (0.10-0.18). Both haplotype Network and the biogeographic analysis showed a structured distribution according to the geographic origin. C. reniformis populations are subdivided into two major clades: Western and Eastern Mediterranean. This pattern seems to be associated with the well-known discontinuous biogeographic area: the Siculo-Tunisian Strait, which separates two water bodies circulating with different hydrological, physical, and chemical characteristics. The short dispersal of pelagic larvae of C. reniformis and the marine bio-geographic barrier created high differentiation among populations. Additionally, it is noteworthy to mention that the "Mahres/Kerkennah" group diverged from Eastern groups in a single sub-clade. This result was expected, the region Mahres/Kerkennah, presented a particular marine environment.

Blocking Toll-like receptor 9 attenuates bleomycin-induced pulmonary injury.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is one of the most common complications in coronavirus disease 2019 patients suffering from acute lung injury (ALI). In ARDS, marked distortion of pulmonary architecture has been reported. The pulmonary lesions in ARDS include hemodynamic derangements (such as alveolar edema and hemorrhage), vascular and bronchiolar damage, interstitial inflammatory cellular aggregations, and eventually fibrosis. Bleomycin induces ARDS-representative pulmonary damage in mice and rats; therefore, we used bleomycin model mice in our study. Recently, Toll-like receptor 9 (TLR9) was implicated in the development of ARDS and ALI. METHODS: In this study, we evaluated the efficiency of a TLR9 blocker (ODN2088) on bleomycin-induced pulmonary damage. We measured the apoptosis rate, inflammatory reaction, and fibroplasia in bleomycin- and bleomycin + ODN2088-treated mice. RESULTS: Our results showed a significant amelioration in bleomycin-induced damage to pulmonary architecture following ODN2088 treatment. A marked decrease in pulmonary epithelial and endothelial apoptosis rate as measured by cleaved caspase-3 expression, inflammatory reaction as indicated by tumor necrosis factor α expression, and pulmonary fibrosis as demonstrated by Van Gieson staining and α-smooth muscle actin immunohistochemistry were observed following ODN2088 treatment. CONCLUSIONS: All these findings indicate that blocking downstream TLR9 signaling could be beneficial in prevention or mitigation of ARDS through hemodynamic derangements, inflammation, apoptosis, and fibrosis.

Antitumor T-cell Immunity Contributes to Pancreatic Cancer Immune Resistance.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. Pancreatic tumors are minimally infiltrated by T cells and are largely refractory to immunotherapy. Accordingly, the role of T-cell immunity in pancreatic cancer has been somewhat overlooked. Here, we hypothesized that immune resistance in pancreatic cancer was induced in response to antitumor T-cell immune responses and that understanding how pancreatic tumors respond to immune attack may facilitate the development of more effective therapeutic strategies. We now provide evidence that T-cell-dependent host immune responses induce a PDAC-derived myeloid mimicry phenomenon and stimulate immune resistance. Three KPC mouse models of pancreatic cancer were used: the mT3-2D (Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre) subcutaneous and orthotopic models, as well as the KP1 (p48-CRE/LSL-Kras/Trp53 flox/flox ) subcutaneous model. KPC cancer cells were grown in immunocompetent and immunodeficient C57BL/6 mice and analyzed to determine the impact of adaptive immunity on malignant epithelial cells, as well as on whole tumors. We found that induced T-cell antitumor immunity, via signal transducer and activator of transcription 1 (STAT1), stimulated malignant epithelial pancreatic cells to induce the expression of genes typically expressed by myeloid cells and altered intratumoral immunosuppressive myeloid cell profiles. Targeting the Janus Kinase (JAK)/STAT signaling pathway using the FDA-approved drug ruxolitinib overcame these tumor-protective responses and improved anti-PD-1 therapeutic efficacy. These findings provide future directions for treatments that specifically disable this mechanism of resistance in PDAC.

The Role of Immunomodulatory Receptors in the Pathogenesis of HIV Infection: A Therapeutic Opportunity for HIV Cure?

Immune activation is the hallmark of HIV infection and plays a role in the pathogenesis of the disease. In the context of suppressed HIV RNA replication by combination antiretroviral therapy (cART), there remains immune activation which is associated to the HIV reservoirs. Persistent virus contributes to a sustained inflammatory environment promoting accumulation of "activated/exhausted" T cells with diminished effector function. These T cells show increased expression of immunomodulatory receptors including Programmed cell death protein (PD1), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA4), Lymphocyte activation gene 3 (LAG3), T cell immunoglobulin and ITIM domain (TIGIT), T cell immunoglobulin and mucin domain containing 3 (TIM3) among others. More importantly, recent reports had demonstrated that, HIV infected T cells express checkpoint receptors, contributing to their survival and promoting maintenance of the viral reservoir. Therapeutic strategies are focused on viral reservoir elimination and/or those to achieve sustained cART-free virologic remission. In this review, we will discuss the immunological basis and the latest advances of the use of checkpoint inhibitors to treat HIV infection.

A single lentivector DNA based immunization contains a late heterologous SIVmac251 mucosal challenge infection.

Variety of conventional vaccine strategies tested against HIV-1 have failed to induce protection against HIV acquisition or durable control of viremia. Therefore, innovative strategies that can induce long lasting protective immunity against HIV chronic infection are needed. Recently, we developed an integration-defective HIV lentiDNA vaccine that undergoes a single cycle of replication in target cells in which most viral antigens are produced. A single immunization with such lentiDNA induced long-lasting T-cell and modest antibody responses in cynomolgus macaques. Here eighteen months after this single immunization, all animals were subjected to repeated low dose intra-rectal challenges with a heterologous pathogenic SIVmac251 isolate. Although the viral set point in SIVmac-infected cynomolgus is commonly lower than that seen in Indian rhesus macaques, the vaccinated group of macaques displayed a two log reduction of peak of viremia followed by a progressive and sustained control of virus replication relative to control animals. This antiviral control correlated with antigen-specific CD4+ and CD8+ T cells with high capacity of recall responses comprising effector and central memory T cells but also memory T cell precursors. This is the first description of SIV control in NHP model infected at 18 months following a single immunization with a non-integrative single cycle lentiDNA HIV vaccine. While not delivering sterilizing immunity, our single immunization strategy with a single-cycle lentivector DNA vaccine appears to provide an interesting and safe vaccine platform that warrants further exploration.

SpCas9-expression by tumor cells can cause T cell-dependent tumor rejection in immunocompetent mice.

The CRISPR/Cas9 system has recently emerged as a highly efficient modality in genetic engineering and has been widely considered for various therapeutic applications. However, since the effector protein, SpCas9, has a bacterial origin, its immunogenicity must be explored in further depth. Here, we found that the intact immune system, in wild-type C57BL/6J and BALB/cL mice, stimulates specific immune response against SpCas9, resulting in the rejection of SpCas9-expressing tumors. However, these tumors effectively grew in syngeneic C57BL/6J immunodeficient, T cell-depleted and Cas9-KI mice. Therefore, these observations suggest that this tumor rejection phenotype is T cell-dependent. The immunological clearance of SpCas9-expressing tumors in the immunocompetent group illustrates the possibility of misinterpreting the impact of CRISPR/Cas9-mediated gene editing on in vivo tumor biology and survival. Thus, these findings have important implications for the use of this exciting approach in in vivo studies, as well as to manipulate cancer cell biology for therapeutic applications.

A novel non-integrative single-cycle chimeric HIV lentivector DNA vaccine.

Novel HIV vaccine vectors and strategies are needed to control HIV/AIDS epidemic in humans and eradicate the infection. DNA vaccines alone failed to induce immune responses robust enough to control HIV-1. Development of lentivirus-based DNA vaccines deficient for integration and with a limited replication capacity is an innovative and promising approach. This type of vaccine mimics the early stages of virus infection/replication like the live-attenuated viruses but lacks the inconvenient integration and persistence associated with disease. We developed a novel lentivector DNA vaccine "CAL-SHIV-IN(-)" that undergoes a single round of replication in the absence of integration resulting in augmented expression of vaccine antigens in vivo. Vaccine gene expression is under control of the LTRs of a naturally attenuated lentivirus, Caprine arthritis encephalitis virus (CAEV) the natural goat lentivirus. The safety of this vaccine prototype was increased by the removal of the integrase coding sequences from the pol gene. We examined the functional properties of this lentivector DNA in cell culture and the immunogenicity in mouse models. Viral proteins were expressed in transfected cells, assembled into viral particles that were able to transduce once target permissive cells. Unlike the parental replication-competent SHIV-KU2 that was detected in DNA samples from any of the serial passage infected cells, CAL-SHIV-IN(-) DNA was detected only in target cells of the first round of infection, hence demonstrating the single cycle replication of the vaccine. A single dose DNA immunization of humanized NOD/SCID/ß2 mice showed a substantial increase of IFN-γ-ELISPOT in splenocytes compared to the former replication and integration defective Δ4SHIV-KU2 DNA vaccine.

Long-term central and effector SHIV-specific memory T cell responses elicited after a single immunization with a novel lentivector DNA vaccine.

Prevention of HIV acquisition and replication requires long lasting and effective immunity. Given the state of HIV vaccine development, innovative vectors and immunization strategies are urgently needed to generate safe and efficacious HIV vaccines. Here, we developed a novel lentivirus-based DNA vector that does not integrate in the host genome and undergoes a single-cycle of replication. Viral proteins are constitutively expressed under the control of Tat-independent LTR promoter from goat lentivirus. We immunized six macaques once only with CAL-SHIV-IN- DNA using combined intramuscular and intradermal injections plus electroporation. Antigen-specific T cell responses were monitored for 47 weeks post-immunization (PI). PBMCs were assessed directly ex vivo or after 6 and 12 days of in vitro culture using antigenic and/or homeostatic proliferation. IFN-γ ELISPOT was used to measure immediate cytokine secretion from antigen specific effector cells and from memory precursors with high proliferative capacity (PHPC). The memory phenotype and functions (proliferation, cytokine expression, lytic content) of specific T cells were tested using multiparametric FACS-based assays. All immunized macaques developed lasting peripheral CD8+ and CD4+ T cell responses mainly against Gag and Nef antigens. During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes. These responses contracted but then reemerged later in absence of antigen boost. Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI. Altogether, our study demonstrated that a single immunization with a replication-limited DNA vaccine elicited persistent vaccine-specific CM and EM CD8+ and CD4+ T cells with immediate and readily inducible effector functions, in the absence of ongoing antigen expression.