Clocks, Viruses, and Immunity: Lessons for the COVID-19 Pandemic.

Circadian rhythms are evolutionarily conserved anticipatory systems that allow the host to prepare and respond to threats in its environment. This article summarizes a European Biological Rhythms Society (EBRS) workshop held in July 2020 to review current knowledge of the interplay between the circadian clock and viral infections to inform therapeutic strategies against SARS-CoV-2 and COVID-19. A large body of work supports the role of the circadian clock in regulating various aspects of viral replication, host responses, and associated pathogenesis. We review the evidence describing the multifaceted role of the circadian clock, spanning host susceptibility, antiviral mechanisms, and host resilience. Finally, we define the most pressing research questions and how our knowledge of chronobiology can inform key translational research priorities.

Carvacrol inhibits the excessive immune response induced by influenza virus A via suppressing viral replication and TLR/RLR pattern recognition.

ETHNOPHARMACOLOGICAL RELEVANCE: Carvacrol, a monoterpene phenol from Mosla chinensis Maxim, which is a commonly Chinese herbal medicine. The most important pharmacology of it is dispelling exogenous evils by increasing perspiration. And it is the gentleman medicine in the Chinese herbal compound prescription of Xin-Jia-Xiang-Ru-Yin, mainly for the treatment of summer colds with dampness including influenza virus A infection. AIM OF THE STUDY: Our preliminary study verified that the Xin-Jia-Xiang-Ru-Yin could inhibit acute lung injury of mice with influenza virus A infection. And there have been some reports implicating the high antimicrobial activity of carvacrol for a wide range of product preservation, but little research including the effects of it on viral infection. The aim of this study was to reveal the antiviral effects of carvacrol, the main constituent in Mosla chinensis Maxim. MATERIALS AND METHODS: Initially, C57BL/6 mice were grouped and intranasally administered FM1 virus to construct viral infection models. After treatment with ribavirin and carvacrol for 5 days, all mice were euthanized, and specimens were immediately obtained. Histology, flow cytometry and Meso Scale Discovery (MSD) analysis were used to analyze pathological changes in lung tissue, the expression levels of cytokines and the differentiation and proportion of CD4+ T cells subsets, while Western blot and qRT-PCR were used to detect the expression of related proteins and mRNA. RESULTS: Carvacrol attenuated lung tissue damage, the proportions of Th1, Th2, Th17 and Treg in CD4+ T cells and the relative proportions of Th1/Th2 and Th17/Treg cells. Carvacrol inhibited the expression of inflammation-associated cytokines including IFN-γ, IL-2, IL-4, IL-5, IL-12 and TNF-ɑ, IL-1, IL-10, IL-6. Decreased levels of TLR7, MyD88, IRAK4, TRAK6, NF-κB, RIG-I, IPS-I and IRF mRNA in carvacrol-treated mice were observed comparing to the mice in VC group. Further, the total expression of RIG-I, MyD88 and NF-κB proteins had increased significantly in the VC group but reduced obviously in the group treated with ribavirin or carvacrol. CONCLUSIONS: These results indicate that carvacrol is a potential alternative treatment for the excessive immune response induced by influenza virus A infection, the cold-fighting effect of Mosla chinensis Maxim may depend on the anti-virus of carvacrol.

Autologous IgG antibodies block outgrowth of a substantial but variable fraction of viruses in the latent reservoir for HIV-1.

In untreated HIV-1 infection, rapid viral evolution allows escape from immune responses. Viral replication can be blocked by antiretroviral therapy. However, HIV-1 persists in a latent reservoir in resting CD4+ T cells, and rebound viremia occurs following treatment interruption. The reservoir, which is maintained in part by clonal expansion, can be measured using quantitative viral outgrowth assays (QVOAs) in which latency is reversed with T cell activation to allow viral outgrowth. Recent studies have shown that viruses detected in QVOAs prior to treatment interruption often differ from rebound viruses. We hypothesized that autologous neutralizing antibodies directed at the HIV-1 envelope (Env) protein might block outgrowth of some reservoir viruses. We modified the QVOA to reflect pressure from low concentrations of autologous antibodies and showed that outgrowth of a substantial but variable fraction of reservoir viruses is blocked by autologous contemporaneous immunoglobulin G (IgG). A reduction in outgrowth of >80% was seen in 6 of 15 individuals. This effect was due to direct neutralization. We established a phylogenetic relationship between rebound viruses and viruses growing out in vitro in the presence of autologous antibodies. Some large infected cell clones detected by QVOA carried neutralization-sensitive viruses, providing a cogent explanation for differences between rebound virus and viruses detected in standard QVOAs. Measurement of the frequency of reservoir viruses capable of outgrowth in the presence of autologous IgG might allow more accurate prediction of time to viral rebound. Ultimately, therapeutic immunization targeting the subset of variants resistant to autologous IgG might contribute to a functional cure.

Can Probiotics and Diet Promote Beneficial Immune Modulation and Purine Control in Coronavirus Infection?

Infection caused by the SARS-CoV-2 coronavirus worldwide has led the World Health Organization to declare a COVID-19 pandemic. Because there is no cure or treatment for this virus, it is emergingly urgent to find effective and validated methods to prevent and treat COVID-19 infection. In this context, alternatives related to nutritional therapy might help to control the infection. This narrative review proposes the importance and role of probiotics and diet as adjunct alternatives among the therapies available for the treatment of this new coronavirus. This review discusses the relationship between intestinal purine metabolism and the use of Lactobacillus gasseri and low-purine diets, particularly in individuals with hyperuricemia, as adjuvant nutritional therapies to improve the immune system and weaken viral replication, assisting in the treatment of COVID-19. These might be promising alternatives, in addition to many others that involve adequate intake of vitamins, minerals and bioactive compounds from food.

AMP-Activated Protein Kinase Restricts Zika Virus Replication in Endothelial Cells by Potentiating Innate Antiviral Responses and Inhibiting Glycolysis.

Viruses are known to perturb host cellular metabolism to enable their replication and spread. However, little is known about the interactions between Zika virus (ZIKV) infection and host metabolism. Using primary human retinal vascular endothelial cells and an established human endothelial cell line, we investigated the role of AMP-activated protein kinase (AMPK), a master regulator of energy metabolism, in response to ZIKV challenge. ZIKV infection caused a time-dependent reduction in the active phosphorylated state of AMPK and of its downstream target acetyl-CoA carboxylase. Pharmacological activation of AMPK using 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), metformin, and a specific AMPKα activator (GSK621) attenuated ZIKV replication. This activity was reversed by an AMPK inhibitor (compound C). Lentivirus-mediated knockdown of AMPK and the use of AMPKα-/- mouse embryonic fibroblasts provided further evidence that AMPK has an antiviral effect on ZIKV replication. Consistent with its antiviral effect, AMPK activation potentiated the expression of genes with antiviral properties (e.g., IFNs, OAS2, ISG15, and MX1) and inhibited inflammatory mediators (e.g., TNF-α and CCL5). Bioenergetic analysis showed that ZIKV infection evokes a glycolytic response, as evidenced by elevated extracellular acidification rate and increased expression of key glycolytic genes (GLUT1, HK2, TPI, and MCT4); activation of AMPK by AICAR treatment reduced this response. Consistent with this, 2-deoxyglucose, an inhibitor of glycolysis, augmented AMPK activity and attenuated ZIKV replication. Thus, our study demonstrates that the anti-ZIKV effect of AMPK signaling in endothelial cells is mediated by reduction of viral-induced glycolysis and enhanced innate antiviral responses.

Effect of high doses of vitamin D supplementation on dengue virus replication, Toll-like receptor expression, and cytokine profiles on dendritic cells.

Dengue, caused by dengue virus (DENV) infection, is a public health problem worldwide. Although DENV pathogenesis has not yet been fully elucidated, the inflammatory response is a hallmark feature in severe DENV infection. Although vitamin D (vitD) can promote the innate immune response against virus infection, no studies have evaluated the effects of vitD on DENV infection, dendritic cells (DCs), and inflammatory response regulation. This study aimed to assess the impact of oral vitD supplementation on DENV-2 infection, Toll-like receptor (TLR) expression, and both pro- and anti-inflammatory cytokine production in monocyte-derived DCs (MDDCs). To accomplish this, 20 healthy donors were randomly divided into two groups and received either 1000 or 4000 international units (IU)/day of vitD for 10 days. During pre- and post-vitD supplementation, peripheral blood samples were taken to obtain MDDCs, which were challenged with DENV-2. We found that MDDCs from donors who received 4000 IU/day of vitD were less susceptible to DENV-2 infection than MDDCs from donors who received 1000 IU/day of vitD. Moreover, these cells showed decreased mRNA expression of TLR3, 7, and 9; downregulation of IL-12/IL-8 production; and increased IL-10 secretion in response to DENV-2 infection. In conclusion, the administration of 4000 IU/day of vitD decreased DENV-2 infection. Our findings support a possible role of vitD in improving the innate immune response against DENV. However, further studies are necessary to determine the role of vitD on DENV replication and its innate immune response modulation in MDDCs.

Mechanisms of Innate Immune Activation by a Hybrid Alphavirus-Rhabdovirus Vaccine Platform.

Virus-like vesicles (VLV) are infectious, self-propagating alphavirus-vesiculovirus hybrid vaccine vectors that can be engineered to express foreign antigens to elicit a protective immune response. VLV are highly immunogenic and nonpathogenic in vivo, and we hypothesize that the unique replication and structural characteristics of VLV efficiently induce an innate antiviral response that enhances immunogenicity and limits replication and spread of the vector. We found that VLV replication is inhibited by interferon (IFN)-α, IFN-γ, and IFN-λ, but not by tumor necrosis factor-α. In cell culture, VLV infection activated IFN production and expression of IFN-stimulated genes (ISGs), such as MXA, ISG15, and IFI27, which were dependent on replication of the evolved VLV-encoded Semliki Forest virus replicon. Knockdown of the pattern recognition receptors, retinoic acid-inducible gene I and melanoma differentiation-associated protein 5 or their intermediary signaling protein mitochondrial antiviral-signaling protein (MAVS) blocked IFN production. Furthermore, ISG expression in VLV-infected cells was dependent on IFN receptor signaling through the Janus kinase (JAK) tyrosine kinases and phosphorylation of the STAT1 protein, and JAK inhibition restored VLV replication in otherwise uninfectable cell lines. This work provides new insight into the mechanism of innate antiviral responses to a hybrid virus-based vector and provides the basis for future characterization of the platform's safety and adjuvant-like effects in vivo. [Figure: see text].

Taishan Pinus massoniana Pollen Polysaccharides Enhance Immune Responses in Chickens Infected by Avian Leukosis Virus Subgroup B.

Immunosuppressive virus, which can cause suppressed immunity and vaccination failure, frequently occurs in chicken flocks and seriously destroys the poultry industry. Our previous studies have reported that Taishan Pinus massoniana pollen polysaccharide (TPPPS) possess immunomodulatory effects and improve the immune effects of vaccines. In this study, avian leukosis virus subgroup B (ALV-B) was chosen as immunosuppressive virus to artificially establish immunosuppressive models in chickens, and the immune modulatory ability of TPPPS on the immune response of chickens was evaluated. Four randomly assigned groups (Group I-IV) of these immunosuppressed chickens were administered with TPPPS at doses of 0, 100, 200, and 400 mg/kg (every kilogram chick), respectively. Group V was administered with saline as control. At seven day old, 10 chickens randomly selected from Group I-V were inoculated with the attenuated Newcastle disease (ND) vaccine. The results showed that during the monitoring period, TPPPS significantly enhanced weight of immune organs, peripheral lymphocyte proliferation, the percentage of CD4+ and the ratio of CD4+/CD8+, IL-2 and IFN-γ production, and ALV-B antibody positive rate of chickens in a dose-dependent manner, with 400 mg/kg TPPPS being the most effective. In addition, the antibody titer against Newcastle disease virus (NDV) in Group IV with 400 mg/kg was significantly higher than those in other groups. We observed the stronger immunity in the TPPPS group, which indicates that TPPPS could be used as an immunoenhancer to relieve immunosuppression caused by ALV-B in the poultry industry.

(-)-Epigallocatechin-3-gallate enhances poly I:C-induced interferon-λ1 production and inhibits hepatitis C virus replication in hepatocytes.

AIM: To investigate the effect of (-)-epigallocatechin-3-gallate (EGCG) on polyinosinic-polycytidylic acid (poly I:C)-triggered intracellular innate immunity against hepatitis C virus (HCV) in hepatocytes. METHODS: A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain (JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular mRNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon (IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells. RESULTS: Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFN-stimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION: Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.

In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers.

Hantaviruses encompass rodent-borne zoonotic pathogens that cause severe hemorrhagic fever disease with high mortality rates in humans. Detection of infectious virus titer lays a solid foundation for virology and immunology researches. Canonical methods to assess viral titers rely on visible cytopathic effects (CPE), but Hantaan virus (HTNV, the prototype hantavirus) maintains a relatively sluggish life cycle and does not produce CPE in cell culture. Here, an in-cell Western (ICW) assay was utilized to rapidly measure the expression of viral proteins in infected cells and to establish a novel approach to detect viral titers. Compared with classical approaches, the ICW assay is accurate and time- and cost-effective. Furthermore, the ICW assay provided a high-throughput platform to screen and identify antiviral molecules. Potential antiviral roles of several DExD/H box helicase family members were investigated using the ICW assay, and the results indicated that DDX21 and DDX60 reinforced IFN responses and exerted anti-hantaviral effects, whereas DDX50 probably promoted HTNV replication. Additionally, the ICW assay was also applied to assess NAb titers in patients and vaccine recipients. Patients with prompt production of NAbs tended to have favorable disease outcomes. Modest NAb titers were found in vaccinees, indicating that current vaccines still require improvements as they cannot prime host humoral immunity with high efficiency. Taken together, our results indicate that the use of the ICW assay to evaluate non-CPE Hantaan virus titer demonstrates a significant improvement over current infectivity approaches and a novel technique to screen antiviral molecules and detect NAb efficacies.

Systems pharmacology uncovers Janus functions of botanical drugs: activation of host defense system and inhibition of influenza virus replication.

Given the imminent threat of influenza pandemics and continuing emergence of new drug-resistant influenza virus strains, novel strategies for preventing and treating influenza disease are urgently needed. Herbal medicine, used for thousands of years in combinational therapies (Herb Formula), plays a significant role in stimulating the host immune system in vivo, and meanwhile, in fighting against the pandemic by directly inhibiting influenza virus in vitro. Such potential Janus functions may spark interest in therapeutic manipulation of virus diseases. Unfortunately, the molecular mechanism of the Janus functions of the medicinal herbs in the treatment of influenza remains unclear. In this work, to illustrate the therapeutic concept of Janus functions in the treatment of influenza, we have introduced a novel systems pharmacology model that integrates pharmacokinetic screening, targeting and network analysis of two representative herbs Lonicera japonica and Fructus Forsythiae that are efficient in the treatment of influenza, inflammation and other diseases. 50 Chemicals with favorable pharmacokinetic profiles have been identified for the two herbs, and the ligand-target network was constructed by complementing the literature-based experimental data deposited in DrugBank. The annotation of these chemicals was assigned using a novel drug targeting approach, and mapped to target-disease and drug-target-pathway networks. The overall data suggest that the medicinal herbs function by indirectly suppressing the virus proliferation via regulating the immune systems in hosts, and also, by directly inhibiting virus proliferation through targeting viral proteins essential for the viral life cycle. For the first time, we have demonstrated the mechanism of medicinal herbs in prevention and treatment of virus diseases via the Janus functions on a systematic level.

Coenzyme Q plays opposing roles on bacteria/fungi and viruses in Drosophila innate immunity.

Coenzyme Q (CoQ or ubiquinone) is a lipid-soluble component of virtually all types of cell membranes and has been shown to play multiple metabolic functions. Several clinical diseases including encephalomyopathy, cerebellar ataxia and isolated myopathy were shown to be associated with CoQ deficiency. However, the role of CoQ in immunity has not been defined. In the present study, we showed that flies defective in CoQ biosynthetic gene coq2 were more susceptible to bacterial and fungal infections, while were more resistant to viruses. We found that Drosophila contained both CoQ9 and CoQ10, and food supplement of CoQ10 could partially rescue the impaired immune functions of coq2 mutants. Surprisingly, wild-type flies fed CoQ10 became more susceptible to viral infection, which suggested that extra caution should be taken when using CoQ10 as a food supplement. We further showed that CoQ was essential for normal induction of anti-microbial peptides and amplification of viruses. Our work determined CoQ content in Drosophila and described its function in immunity for the first time.

HIV sensitivity to neutralization is determined by target and virus producer cell properties.

OBJECTIVE: Using clinical isolates from a recent passive immunization trial with antibody 2G12, we probed the capacity of frequently used neutralization formats - the primary peripheral blood mononuclear cell (PBMC)-based and the TZM-bl cell-based assay systems - to predict in-vivo activity of 2G12. DESIGN: Antibodies and entry inhibitors of established efficacy were used to neutralize HIV-1 isolates in different in-vitro assay setups. METHODS: Single round infection with Env-pseudotyped and multiple round infection with replication-competent virus was studied on PBMCs and a variety of engineered cell lines. RESULTS: Six out of 12 isolates with high sensitivity to 2G12 in the replication-competent PBMC assay lacked sensitivity to the monoclonal antibody in the env-pseudotype TZM-bl assay. Outcome of passive immunization with 2G12 corroborated the PBMC-assay in-vitro data, as escape mutations to 2G12 emerged, proving the monoclonal antibody's impact on HIV in vivo. Failure to inhibit pseudotype infection of TZM-bl was not due to sequence differences or the pseudotype infection per se, as infection of PBMCs with the identical pseudotyped viruses was sensitive to 2G12 inhibition. Similar shifts in efficacy, though less extreme, were noted for other neutralizing antibodies and inhibitors. Exploration of causes for the observed differences between assay systems revealed that both target cell and virus producer properties influence sensitivity of virus entry to inhibition. CONCLUSION: Our observation that neutralization assay systems employing engineered reporter cell lines can miss in-vivo relevant neutralizing activities strongly argues that preclinical assessment should not be restricted to a single assay type.

Nanochemistry-based immunotherapy for HIV-1.

Highly active antiretroviral treatment (HAART), i.e. the combination of three or more drugs against human immunodeficiency virus type 1 (HIV-1), has greatly improved the clinical outcome of HIV-1-infected individuals. However, HAART is unable to reconstitute HIV-specific immunity and eradicate the virus. Several observations in primate models and in humans support the notion that cell-mediated immunity can control viral replication and slow disease progression. Thus, besides drugs, an immunotherapy that induces long-lasting HIV-specific T-cell responses could play a role in the treatment of HIV/AIDS. To induce such immune responses, DermaVir Patch has been developed. DermaVir consists of an HIV-1 antigen-encoding plasmid DNA that is chemically formulated in a nanoparticle. DermaVir is administered under a patch after a skin preparation that supports the delivery of the nanoparticle to Langerhans cells (LC). Epidermal LC trap and transport the nanomedicine to draining lymph nodes. While in transit, LC mature into dendritic cells (DC), which can efficiently present the DNA-encoded antigens to naïve T-cells for the induction of cellular immunity. Pre-clinical studies and Phase I clinical testing of DermaVir in HIV-1-infected individuals have demonstrated the safety and tolerability of DermaVir Patch. To further modulate cellular immunity, molecular adjuvants might be added into the nanoparticle. DermaVir Patch represents a new nanomedicine platform for immunotherapy of HIV/AIDS. In this review, the antiviral activity of DermaVir-induced cellular immunity is discussed. Furthermore, the action of some cytokines currently being tested as adjuvants are highlighted and the adjuvant effect of cytokine plasmid DNA included in the DermaVir nanoparticle is reviewed.

Dehydroepiandrosterone (DHEA) effects on HIV replication and host immunity: a randomized placebo-controlled study.

Prior studies have indicated that dehydroepiandrosterone (DHEA) may have immunomodulatory properties as well as positive effects on mood, quality of life, and body composition. Preliminary data suggest that DHEA inhibits expression of human immunodeficiency virus 1 (HIV) in latently infected cells; thus, it might be a potential adjunct to currently available antiretroviral therapy. The objective was to determine DHEA's impact on latent HIV infection, persistent viral replication, immunity, and nonimmune aspects of health restoration. A randomized, double-blind, placebo-controlled 24-week outpatient intervention included 40 subjects with suppressed HIV viremia on a stable antiretroviral regimen. Participants were randomized with equal probability to receive either DHEA or placebo for 12 weeks, followed by open-label DHEA for an additional 12 weeks. Intensive virologic monitoring included plasma viral load assays (lower limits of detection 50 copies/ml and 2.5 copies/ml) and quantitative cultures of replication-competent virus reservoirs in blood cells. A full battery of immunologic measurements was performed. Measurements of hormones, body weight, and body composition were obtained. Quality of life was assessed using validated questionnaires. DHEA was bioavailable as ascertained by increased levels of DHEA, DHEA(S), and androstenedione in recipients' plasma compared to the control group. The titers of infectious HIV culturable from blood trended upward in the DHEA arm although there was no significant change in plasma HIV RNA level. No significant immune effects were observed with DHEA. There appeared to be no benefit with regard to lean muscle mass or bone density in the DHEA recipients. DHEA treatment had a positive impact on overall quality of life. DHEA supplementation in fully suppressed HIV patients was associated with an improvement in quality of life but appeared to have no beneficial antiviral, immunomodulatory, hormonal, or body composition effects, suggesting that it not be routinely used as an adjunctive therapy in this population.

AM3 inhibits HBV replication through activation of peripheral blood mononuclear cells.

In this report, we have analyzed the effect of AM3, a glycoconjugate of natural origin with immunomodulatory properties, which is available under the commercial name of Inmunoferon, on hepatitis B virus (HBV) replication in HBV-transfected cells. We found that AM3 inhibited HBV RNA expression as well as DNA synthesis and viral antigen expression by an indirect mechanism. We found that AM3 lacked intrinsic antiviral properties, and that the antiviral effect of the glycoconjugate was due to stimulation of secretion of molecules with antiviral properties by peripheral blood mononuclear cells. Our data indicate that the employment of AM3 as an adjuvant administered simultaneously with conventional antiviral drugs may potentiate the endogenous response against viral infection.

IL-7 stimulates T cell renewal without increasing viral replication in simian immunodeficiency virus-infected macaques.

The main failure of antiretroviral therapy is the lack of restoration of HIV-specific CD4(+) T cells. IL-7, which has been shown to be a crucial cytokine for thymopoiesis, has been envisaged as an additive therapeutic strategy. However, in vitro studies suggest that IL-7 might sustain HIV replication in thymocytes and T lymphocytes. Therefore, in the present study, we evaluated the effect of IL-7 on both T cell renewal and viral load in SIVmac-infected young macaques in the absence of antiretroviral therapy. This evaluation was conducted during the asymptomatic phase in view of a potential treatment of HIV patients. We show that IL-7 induces both a central renewal and a peripheral expansion of T lymphocytes associated with cell activation. No alarming modulation of the other hemopoietic cells was observed. No increase in the viral load was shown in blood or lymph nodes. These data strengthen the rationale for the use of IL-7 as an efficient immunotherapy in AIDS.

Phosphatidylserine on HIV envelope is a cofactor for infection of monocytic cells.

HIV-1 is an enveloped retrovirus that acquires its outer membrane as the virion exits the cell. Because of the association of apoptosis with the progression of AIDS, HIV-1-infected T cells or macrophages might be expected to express elevated levels of surface phosphatidylserine (PS), a hallmark of programmed cell death. Virions produced by these cells would also be predicted to have PS on the surface of their envelopes. In this study, data are presented that support this hypothesis and suggest that PS is required for macrophage infection. The PS-specific protein annexin V was used to enrich for virus particles and to inhibit HIV-1 replication in primary macrophages, but not T cells. HIV-1 replication was also significantly inhibited with vesicles consisting of PS, but not phosphatidylcholine. PS is specifically required for HIV-1 infection because viruses pseudotyped with vesicular stomatitis virus G and amphotropic murine leukemia virus envelopes were not inhibited by PS vesicles or annexin V. These data indicate that PS is an important cofactor for HIV-1 infection of macrophages.

Development of an animal model for autotransfusion therapy: in vitro characterization and analysis of anti-CD3/CD28 expanded cells.

Previous studies have shown that in vitro culture of human CD4+ T cells with antibodies to CD3 and CD28 immobilized on beads induced an antiviral effect to HIV-1 infection. Herein, we have used CD4+ T cells from nonhuman primates to address issues critical for use of such cells for therapy and immune reconstitution of humans and nonhuman primates infected with HIV and simian immunovirus (SIV). These studies include definition of the kinetics of the antiviral effect, the relative stability of the acquired phenotype, and whether such activated and expanded CD4+ T cells retain their immune function. Results of our studies show that antiviral effect is induced rapidly following activation with anti-CD3/CD28-coated beads. Additionally, the antiviral effect is not stable in these cells and requires continuous culture with anti-CD3/CD28 beads. Removal of CD4+ T cells from anti-CD3/CD28 stimulation renders these cells susceptible to infection, demonstrating that the resistant phenotype is not stable in these cultures. However, anti-CD3/CD28 expanded CD4+ T cells do retain immune function. Thus, although these findings imply a note of caution for therapeutic strategies aimed at providing patients with virus-resistant CD4+ T cells, the present study suggests that transfusion of such cells with retained immune function may have immune restoration capability.

The Ick protein tyrosine kinase is not involved in antibody-mediated CD4 (CDR3-loop) signal transduction that inhibits HIV-1 transcription.

Monoclonal antibodies (mAb) that bind to the immunoglobulin CDR3-like region in the D1 domain of the CD4 molecule can inhibit the HIV-1 life cycle in CD4-positive T cells and lymphoblastoid cell lines at the stage of transcription. This antiviral effect requires the integrity of the cytoplasmic tail of CD4 which is known to act as a signal transduction region through its association with the protein tyrosine kinase (PTK) p56lck. In this study, we investigated the putative role of this PTK in transducing inhibitory signals that act on HIV-1 replication after triggering by anti-CDR3-like region antibody treatment of infected T cell lines. CEM (CD4+/p56lck + inducible), MT2 (CD4+/p56lck - repressed), HSB-2 (CD4-/p56lck + constitutively), HSB-2 WTCD4 (CD4+/p56lck + constitutively), HSB-2 CD4.402 (CD4+ truncated form which lacks the cytoplasmic domain/p56lck + constitutively), and HSB-2 CD4mut (CD4+ unable to bind lck/p56lck + constitutively) were exposed to HIV-1 and cultured in medium supplemented with an anti-CDR3-like region-specific antibody or a control anti-CD4 mAb which does not inhibit HIV-1 transcription. We found that CDR3-loop-mediated inhibitory signals are efficiently transduced in CD4-positive cells which demonstrate a constitutive activation of p56lck or in CD4-positive cells lacking p56lck expression. Moreover, inhibitory signals were transduced in HSB-2 CD4mut cells expressing a cell surface CD4 with a double cysteine mutation in its cytoplasmic tail that renders the molecule unable to bind p56lck, but not HSB-2 CD4.402 cells expressing a truncated form of CD4 which lacks the cytoplasmic domain. These results indicate that the p56lck plays no direct role in this process and suggests the existence of another signaling partner for CD4.