Case Study: Prolonged Infectious SARS-CoV-2 Shedding from an Asymptomatic Immunocompromised Individual with Cancer.

Long-term severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shedding was observed from the upper respiratory tract of a female immunocompromised individual with chronic lymphocytic leukemia and acquired hypogammaglobulinemia. Shedding of infectious SARS-CoV-2 was observed up to 70 days, and of genomic and subgenomic RNA up to 105 days, after initial diagnosis. The infection was not cleared after the first treatment with convalescent plasma, suggesting a limited effect on SARS-CoV-2 in the upper respiratory tract of this individual. Several weeks after a second convalescent plasma transfusion, SARS-CoV-2 RNA was no longer detected. We observed marked within-host genomic evolution of SARS-CoV-2 with continuous turnover of dominant viral variants. However, replication kinetics in Vero E6 cells and primary human alveolar epithelial tissues were not affected. Our data indicate that certain immunocompromised individuals may shed infectious virus longer than previously recognized. Detection of subgenomic RNA is recommended in persistently SARS-CoV-2-positive individuals as a proxy for shedding of infectious virus.

Chromothriptic cure of WHIM syndrome.

Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here, we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4(R334X), as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patient's cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.

Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2.

Effective therapies to treat coronavirus disease 2019 (COVID-19) are urgently needed. While many investigational, approved, and repurposed drugs have been suggested as potential treatments, preclinical data from animal models can guide the search for effective treatments by ruling out those that lack efficacy in vivo. Remdesivir (GS-5734) is a nucleotide analogue prodrug with broad antiviral activity1,2 that is currently being investigated in COVID-19 clinical trials and recently received Emergency Use Authorization from the US Food and Drug Administration3,4. In animal models, remdesivir was effective against infection with Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV)2,5,6. In vitro, remdesivir inhibited replication of SARS-CoV-27,8. Here we investigate the efficacy of remdesivir in a rhesus macaque model of SARS-CoV-2 infection9. Unlike vehicle-treated animals, macaques treated with remdesivir did not show signs of respiratory disease; they also showed reduced pulmonary infiltrates on radiographs and reduced virus titres in bronchoalveolar lavages twelve hours after the first dose. Virus shedding from the upper respiratory tract was not reduced by remdesivir treatment. At necropsy, remdesivir-treated animals had lower lung viral loads and reduced lung damage. Thus, treatment with remdesivir initiated early during infection had a clinical benefit in rhesus macaques infected with SARS-CoV-2. Although the rhesus macaque model does not represent the severe disease observed in some patients with COVID-19, our data support the early initiation of remdesivir treatment in patients with COVID-19 to prevent progression to pneumonia.

Modeling Heartland virus disease in mice and therapeutic intervention with 4'-fluorouridine.

Heartland virus (HRTV) is an emerging tick-borne bandavirus that causes a febrile illness of varying severity in humans, with cases reported in eastern and midwestern regions of the United States. No vaccines or approved therapies are available to prevent or treat HRTV disease. Here, we describe the genetic changes, natural history of disease, and pathogenesis of a mouse-adapted HRTV (MA-HRTV) that is uniformly lethal in 7- to 8-week-old AG129 mice at low challenge doses. We used this model to assess the efficacy of the ribonucleoside analog, 4'-fluorouridine (EIDD-2749), and showed that once-daily oral treatment with 3 mg/kg of drug, initiated after the onset of disease, protects mice against lethal MA-HRTV challenge and reduces viral loads in blood and tissues. Our findings provide insights into HRTV virulence and pathogenesis and support further development of EIDD-2749 as a therapeutic intervention for HRTV disease. IMPORTANCE: More than 60 cases of HRTV disease spanning 14 states have been reported to the United States Centers for Disease Control and Prevention. The expanding range of the Lone Star tick that transmits HRTV, the growing population of at-risk persons living in geographic areas where the tick is abundant, and the lack of antiviral treatments or vaccines raise significant public health concerns. Here, we report the development of a new small-animal model of lethal HRTV disease to gain insight into HRTV pathogenesis and the application of this model for the preclinical development of a promising new antiviral drug candidate, EIDD-2749. Our findings shed light on how the virus causes disease and support the continued development of EIDD-2749 as a therapeutic for severe cases of HRTV infection.

Pathogenesis and outcome of VA1 astrovirus infection in the human brain are defined by disruption of neural functions and imbalanced host immune responses.

Astroviruses (AstVs) can cause of severe infection of the central nervous system (CNS) in immunocompromised individuals. Here, we identified a human AstV of the VA1 genotype, HAstV-NIH, as the cause of fatal encephalitis in an immunocompromised adult. We investigated the cells targeted by AstV, neurophysiological changes, and host responses by analyzing gene expression, protein expression, and cellular morphology in brain tissue from three cases of AstV neurologic disease (AstV-ND). We demonstrate that neurons are the principal cells targeted by AstV in the brain and that the cerebellum and brainstem have the highest burden of infection. Detection of VA1 AstV in interconnected brain structures such as thalamus, deep cerebellar nuclei, Purkinje cells, and pontine nuclei indicates that AstV may spread between connected neurons transsynaptically. We found transcriptional dysregulation of neural functions and disruption of both excitatory and inhibitory synaptic innervation of infected neurons. Importantly, transcriptional dysregulation of neural functions occurred in fatal cases, but not in a patient that survived AstV-ND. We show that the innate, but not adaptive immune response was transcriptionally driving host defense in the brain of immunocompromised patients with AstV-ND. Both transcriptome and molecular pathology studies showed that most of the cellular changes were associated with CNS-intrinsic cells involved in phagocytosis and injury repair (microglia, perivascular/parenchymal border macrophages, and astrocytes), but not CNS-extrinsic cells (T and B cells), suggesting an imbalance of innate and adaptive immune responses to AstV infection in the brain as a result of the underlying immunodeficiencies. These results show that VA1 AstV infection of the brain in immunocompromised humans is associated with imbalanced host defense responses, disruption of neuronal somatodendritic compartments and synapses and increased phagocytic cellular activity. Improved understanding of the response to viral infections of the human CNS may provide clues for how to manipulate these processes to improve outcomes.

MAdCAM-1 costimulation in the presence of retinoic acid and TGF-ß promotes HIV infection and differentiation of CD4+ T cells into CCR5+ TRM-like cells.

CD4+ tissue resident memory T cells (TRMs) are implicated in the formation of persistent HIV reservoirs that are established during the very early stages of infection. The tissue-specific factors that direct T cells to establish tissue residency are not well defined, nor are the factors that establish viral latency. We report that costimulation via MAdCAM-1 and retinoic acid (RA), two constituents of gut tissues, together with TGF-ß, promote the differentiation of CD4+ T cells into a distinct subset α4ß7+CD69+CD103+ TRM-like cells. Among the costimulatory ligands we evaluated, MAdCAM-1 was unique in its capacity to upregulate both CCR5 and CCR9. MAdCAM-1 costimulation rendered cells susceptible to HIV infection. Differentiation of TRM-like cells was reduced by MAdCAM-1 antagonists developed to treat inflammatory bowel diseases. These finding provide a framework to better understand the contribution of CD4+ TRMs to persistent viral reservoirs and HIV pathogenesis.

A single intranasal dose of a live-attenuated parainfluenza virus-vectored SARS-CoV-2 vaccine is protective in hamsters.

Single-dose vaccines with the ability to restrict SARS-CoV-2 replication in the respiratory tract are needed for all age groups, aiding efforts toward control of COVID-19. We developed a live intranasal vector vaccine for infants and children against COVID-19 based on replication-competent chimeric bovine/human parainfluenza virus type 3 (B/HPIV3) that express the native (S) or prefusion-stabilized (S-2P) SARS-CoV-2 S spike protein, the major protective and neutralization antigen of SARS-CoV-2. B/HPIV3/S and B/HPIV3/S-2P replicated as efficiently as B/HPIV3 in vitro and stably expressed SARS-CoV-2 S. Prefusion stabilization increased S expression by B/HPIV3 in vitro. In hamsters, a single intranasal dose of B/HPIV3/S-2P induced significantly higher titers compared to B/HPIV3/S of serum SARS-CoV-2-neutralizing antibodies (12-fold higher), serum IgA and IgG to SARS-CoV-2 S protein (5-fold and 13-fold), and IgG to the receptor binding domain (10-fold). Antibodies exhibited broad neutralizing activity against SARS-CoV-2 of lineages A, B.1.1.7, and B.1.351. Four weeks after immunization, hamsters were challenged intranasally with 104.5 50% tissue-culture infectious-dose (TCID50) of SARS-CoV-2. In B/HPIV3 empty vector-immunized hamsters, SARS-CoV-2 replicated to mean titers of 106.6 TCID50/g in lungs and 107 TCID50/g in nasal tissues and induced moderate weight loss. In B/HPIV3/S-immunized hamsters, SARS-CoV-2 challenge virus was reduced 20-fold in nasal tissues and undetectable in lungs. In B/HPIV3/S-2P-immunized hamsters, infectious challenge virus was undetectable in nasal tissues and lungs; B/HPIV3/S and B/HPIV3/S-2P completely protected against weight loss after SARS-CoV-2 challenge. B/HPIV3/S-2P is a promising vaccine candidate to protect infants and young children against HPIV3 and SARS-CoV-2.

Atypical Ebola Virus Disease in a Rhesus Macaque.

Ebola virus (EBOV)-Makona infected more than 30 000 people from 2013 to 2016 in West Africa, among them many health care workers including foreign nationals. Most of the infected foreign nationals were evacuated and treated in their respective home countries, resulting in detailed reports of the acute disease following EBOV infection as well as descriptions of symptoms now known as post-Ebola syndrome, which occurred months after the infection. Symptoms associated with this syndrome include uveitis and neurological manifestations. In 1 of our EBOV-Makona nonhuman primate (NHP) studies, 1 NHP was euthanized on day 28 after infection having completely recovered from the acute disease. During convalescence, this NHP developed neurological signs and acute respiratory distress requiring euthanasia. The organ tropism had changed with high virus titers in lungs, brain, eye, and reproductive organs but no virus in the typical target organs for acute EBOV infection. This in part reflects sequelae described for EBOV survivors albeit developing quicker after recovery from acute disease.

TargeTron Inactivation of Chlamydia trachomatis gseA Results in a Lipopolysaccharide 3-Deoxy-d-Manno-Oct-2-Ulosonic Acid-Deficient Strain That Is Cytotoxic for Cells.

All members of the family Chlamydiaceae have lipopolysaccharides (LPS) that possess a shared carbohydrate trisaccharide antigen, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) that is functionally uncharacterized. A single gene, genus-specific epitope (gseA), is responsible for attaching the tri-Kdo to lipid IVA. To investigate the function of Kdo in chlamydial host cell interactions, we made a gseA-null strain (L2ΔgseA) by using TargeTron mutagenesis. Immunofluorescence microscopy and immunoblotting with a Kdo-specific monoclonal antibody demonstrated that L2ΔgseA lacked Kdo. L2ΔgseA reacted by immunoblotting with a monoclonal antibody specific for a conserved LPS glucosamine-PO4 epitope, indicating that core lipid A was retained by the mutant. The mutant strain produced a similar number of inclusions as the parental strain but yielded lower numbers of infectious elementary bodies. Transmission electron microscopy of L2ΔgseA-infected cells showed atypical developmental forms and a reduction in the number of elementary bodies. Immunoblotting of dithiothreitol-treated L2ΔgseA-infected cells lysates revealed a marked reduction in outer membrane OmcB disulfide cross-linking, suggesting that the elementary body outer membrane structure was affected by the lack of Kdo. Notably, lactic acid dehydrogenase release by infected cells demonstrated that L2ΔgseA was significantly more cytotoxic to host cells than the wild type. The cytotoxic phenotype may result from an altered outer membrane biogenesis structure and/or function or, conversely, from a direct pathobiological effect of Kdo on an unknown host cell target. These findings implicate a previously unrecognized role for Kdo in host cell interactions that facilitates postinfection host cell survival.

Zombie cats on the quantum-classical frontier: Wigner-Moyal and semiclassical limit dynamics of quantum coherence in molecules.

In this paper, we investigate the time evolution of quantum coherence-the off-diagonal elements of the density matrix of a multistate quantum system-from the perspective of the Wigner-Moyal formalism. This approach provides an exact phase space representation of quantum mechanics. We consider the coherent evolution of nuclear wavepackets in a molecule with two electronic states. For harmonic potentials, the problem is analytically soluble for both a fully quantum mechanical description and a semiclassical description. We highlight the serious deficiencies of the semiclassical treatment of coherence for general systems and illustrate how even qualitative accuracy requires higher order terms in the Moyal expansion to be included. The model provides an experimentally relevant example of a molecular Schrödinger's cat state. The alive and dead cats of the exact two-state quantum evolution collapse into a "zombie" cat in the semiclassical limit-an averaged behavior, neither alive nor dead, leading to significant errors. The inclusion of the Moyal correction restores a faithful simultaneously alive and dead representation of the cat that is experimentally observable.

A first principles derivation of energy-conserving momentum jumps in surface hopping simulations.

The fewest switches surface hopping (FSSH) method proposed by Tully in 1990 [Tully, J. Chem. Phys. 93, 1061 (1990)]-along with its many later variations-forms the basis for most practical simulations of molecular dynamics with electronic transitions in realistic systems. Despite its popularity, a rigorous formal derivation of the algorithm has yet to be achieved. In this paper, we derive the energy-conserving momentum jumps employed by FSSH from the perspective of quantum trajectory surface hopping (QTSH) [Martens, J. Phys. Chem. A 123, 1110 (2019)]. In the limit of localized nonadiabatic transitions, simple mathematical and physical arguments allow the FSSH algorithm to be derived from first principles. For general processes, the quantum forces characterizing the QTSH method provide accurate results for nonadiabatic dynamics with rigorous energy conservation, at the ensemble level, within the consistency of the underlying stochastic surface hopping without resorting to the artificial momentum rescaling of FSSH.

Posttranscriptional regulation of human endogenous retroviruses by RNA-binding motif protein 4, RBM4.

The human genome encodes for over 1,500 RNA-binding proteins (RBPs), which coordinate regulatory events on RNA transcripts. Most studies of RBPs have concentrated on their action on host protein-encoding mRNAs, which constitute a minority of the transcriptome. A widely neglected subset of our transcriptome derives from integrated retroviral elements, termed endogenous retroviruses (ERVs), that comprise ∼8% of the human genome. Some ERVs have been shown to be transcribed under physiological and pathological conditions, suggesting that sophisticated regulatory mechanisms to coordinate and prevent their ectopic expression exist. However, it is unknown how broadly RBPs and ERV transcripts directly interact to provide a posttranscriptional layer of regulation. Here, we implemented a computational pipeline to determine the correlation of expression between individual RBPs and ERVs from single-cell or bulk RNA-sequencing data. One of our top candidates for an RBP negatively regulating ERV expression was RNA-binding motif protein 4 (RBM4). We used photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation to demonstrate that RBM4 indeed bound ERV transcripts at CGG consensus elements. Loss of RBM4 resulted in an elevated transcript level of bound ERVs of the HERV-K and -H families, as well as increased expression of HERV-K envelope protein. We pinpointed RBM4 regulation of HERV-K to a CGG-containing element that is conserved in the LTRs of HERV-K-10, -K-11, and -K-20, and validated the functionality of this site using reporter assays. In summary, we systematically identified RBPs that may regulate ERV function and demonstrate a role for RBM4 in controlling ERV expression.

Transcriptome profiling of Plasmodium vivax in Saimiri monkeys identifies potential ligands for invasion.

Unlike the case in Asia and Latin America, Plasmodium vivax infections are rare in sub-Saharan Africa due to the absence of the Duffy blood group antigen (Duffy antigen), the only known erythrocyte receptor for the P. vivax merozoite invasion ligand, Duffy binding protein 1 (DBP1). However, P. vivax infections have been documented in Duffy-negative individuals throughout Africa, suggesting that P. vivax may use ligands other than DBP1 to invade Duffy-negative erythrocytes through other receptors. To identify potential P. vivax ligands, we compared parasite gene expression in Saimiri and Aotus monkey erythrocytes infected with P. vivax Salvador I (Sal I). DBP1 binds Aotus but does not bind to Saimiri erythrocytes; thus, P. vivax Sal I must invade Saimiri erythrocytes independent of DBP1. Comparing RNA sequencing (RNAseq) data for late-stage infections in Saimiri and Aotus erythrocytes when invasion ligands are expressed, we identified genes that belong to tryptophan-rich antigen and merozoite surface protein 3 (MSP3) families that were more abundantly expressed in Saimiri infections compared with Aotus infections. These genes may encode potential ligands responsible for P. vivax infections of Duffy-negative Africans.

Similar Frequency and Inducibility of Intact Human Immunodeficiency Virus-1 Proviruses in Blood and Lymph Nodes.

BACKGROUND: The human immunodeficiency virus (HIV)-1 latent reservoir (LR) in resting CD4+ T cells is a barrier to cure. LR measurements are commonly performed on blood samples and therefore may miss latently infected cells residing in tissues, including lymph nodes. METHODS: We determined the frequency of intact HIV-1 proviruses and proviral inducibility in matched peripheral blood (PB) and lymph node (LN) samples from 10 HIV-1-infected patients on antiretroviral therapy (ART) using the intact proviral DNA assay and a novel quantitative viral induction assay. Prominent viral sequences from induced viral RNA were characterized using a next-generation sequencing assay. RESULTS: The frequencies of CD4+ T cells with intact proviruses were not significantly different in PB versus LN (61/106 vs 104/106 CD4+ cells), and they were substantially lower than frequencies of CD4+ T cells with defective proviruses. The frequencies of CD4+ T cells induced to produce high levels of viral RNA were not significantly different in PB versus LN (4.3/106 vs 7.9/106), but they were 14-fold lower than the frequencies of cells with intact proviruses. Sequencing of HIV-1 RNA from induced proviruses revealed comparable sequences in paired PB and LN samples. CONCLUSIONS: These results further support the use of PB as an appropriate proxy for the HIV-1 LR in secondary lymphoid organs.

Longitudinal Antibody Responses in People Who Inject Drugs Infected With Similar Human Immunodeficiency Virus Strains.

BACKGROUND: Multiple factors influence the human immunodeficiency virus (HIV) antibody response produced during natural infection, leading to responses that can vary in specificity, strength, and breadth. METHODS: People who inject drugs identified as recently infected with HIV (n = 23) were analyzed for clustering of their viral sequences (genetic distance, <2%). Longitudinal antibody responses were identified for neutralizing antibody (Nab) potential, and differences in antibody subclass, specificity, and Fc receptor ligation using pseudovirus entry and multiplexed Fc array assays, respectively. Responses were analyzed for differences between subject groups, defined by similarity in the sequence of the infecting virus. RESULTS: Viral sequences from infected individuals were grouped into 3 distinct clusters with 7 unclustered individuals. Subjects in cluster 1 generally had lower antibody response magnitudes, except for antibodies targeting the V1/V2 region. Subjects in clusters 2 and 3 typically had higher antibody response magnitudes, with the Fv specificity of cluster 2 favoring gp140 recognition. NAb responses differed significantly between clusters for 3 of 18 pseudoviruses examined (P < .05), but there were no differences in overall NAb breadth (P = .62). DISCUSSION: These data demonstrate that individuals infected with similar viral strains can generate partially similar antibody responses, but these do not drastically differ from those in individuals infected with relatively unrelated strains.

Antiretroviral Adherence, Elevated Viral Load, and Drug Resistance Mutations in Human Immunodeficiency Virus-infected Women Initiating Treatment in Pregnancy: A Nested Case-control Study.

BACKGROUND: Elevated viral load (VL) early after antiretroviral therapy (ART) initiation appears frequently in pregnant and postpartum women living with human immunodeficiency virus; however the relative contributions of pre-ART drug resistance mutations (DRMs) vs nonadherence in the etiology of elevated VL are unknown. METHODS: Within a cohort of women initiating ART during pregnancy in Cape Town, South Africa, we compared women with elevated VL after initial suppression (cases, n = 80) incidence-density matched to women who maintained suppression over time (controls, n = 87). Groups were compared on pre-ART DRMs and detection of antiretrovirals in stored plasma. RESULTS: The prevalence of pre-ART DRMs was 10% in cases and 5% in controls (adjusted odds ratio [aOR], 1.53 [95% confidence interval {CI}, .4-5.9]); all mutations were to nonnucleoside reverse transcriptase inhibitors. At the time of elevated VL, 19% of cases had antiretrovirals detected in plasma, compared with 87% of controls who were suppressed at a matched time point (aOR, 131.43 [95% CI, 32.8-527.4]). Based on these findings, we estimate that <10% of all elevated VL in the cohort may be attributable to pre-ART DRMs vs >90% attributable to ART nonadherence. CONCLUSIONS: DRMs account for a small proportion of all elevated VL among women occurring in the 12 months after ART initiation during pregnancy in this setting, with nonadherence appearing to drive most episodes of elevated VL. Alongside the drive for access to more robust antiretroviral agents in resource-limited settings, there is an ongoing need for effective strategies to support ART adherence in this patient population.

Helios+ and Helios- Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires.

The transcription factor Helios is expressed in a large subset of Foxp3+ Tregs. We previously proposed that Helios is a marker of thymic derived Treg (tTreg), while Helios- Treg were induced from Foxp3- T conventional (Tconv) cells in the periphery (pTreg). To compare the two Treg subpopulations, we generated Helios-GFP reporter mice and crossed them to Foxp3-RFP reporter mice. The Helios+ Treg population expressed a more activated phenotype, had a slightly higher suppressive capacity in vitro and expressed a more highly demethylated TSDR but were equivalent in their ability to suppress inflammatory bowel disease in vivo. However, Helios+ Treg more effectively inhibited the proliferation of activated, autoreactive splenocytes from scurfy mice. When Helios+ and Helios- Treg were transferred to lymphoreplete mice, both populations maintained comparable Foxp3 expression, but Foxp3 expression was less stable in Helios- Treg when transferred to lymphopenic mice. Gene expression profiling demonstrated a large number of differentially expressed genes and showed that Helios- Treg expressed certain genes normally expressed in CD4+ Foxp3- T cells. TCR repertoire analysis indicated very little overlap between Helios+ and Helios- Treg. Thus, Helios+ and Helios- Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires.

Lack of Activation Marker Induction and Chemokine Receptor Switch in Human Neonatal Myeloid Dendritic Cells in Response to Human Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) infects and causes disease in infants and reinfects with reduced disease throughout life without significant antigenic change. In contrast, reinfection by influenza A virus (IAV) largely requires antigenic change. The adaptive immune response depends on antigen presentation by dendritic cells (DC), which may be too immature in young infants to induce a fully protective immune response against RSV reinfections. We therefore compared the ability of RSV and IAV to activate primary human cord blood (CB) and adult blood (AB) myeloid DC (mDC). While RSV and IAV infected with similar efficiencies, RSV poorly induced maturation and cytokine production in CB and AB mDC. This difference between RSV and IAV was more profound in CB mDC. While IAV activated CB mDC to some extent, RSV did not induce CB mDC to increase the maturation markers CD38 and CD86 or CCR7, which directs DC migration to lymphatic tissue. Low CCR7 surface expression was associated with high expression of CCR5, which keeps DC in inflamed peripheral tissues. To evaluate a possible inhibition by RSV, we subjected RSV-inoculated AB mDC to secondary IAV inoculation. While RSV-inoculated AB mDC responded to secondary IAV inoculation by efficiently upregulating activation markers and cytokine production, IAV-induced CCR5 downregulation was slightly inhibited in cells exhibiting robust RSV infection. Thus, suboptimal stimulation and weak and mostly reversible inhibition seem to be responsible for inefficient mDC activation by RSV. The inefficient mDC stimulation and immunological immaturity in young infants may contribute to reduced immune responses and incomplete protection against RSV reinfection.IMPORTANCE Respiratory syncytial virus (RSV) causes disease early in life and can reinfect symptomatically throughout life without undergoing significant antigenic change. In contrast, reinfection by influenza A virus (IAV) requires antigenic change. The adaptive immune response depends on antigen presentation by dendritic cells (DC). We used myeloid DC (mDC) from cord blood and adult blood donors to evaluate whether immunological immaturity contributes to the inability to mount a fully protective immune response to RSV. While IAV induced some activation and chemokine receptor switching in cord blood mDC, RSV did not. This appeared to be due to a lack of activation and a weak and mostly reversible inhibition of DC functions. Both viruses induced a stronger activation of mDC from adults than mDC from cord blood. Thus, inefficient stimulation of mDC by RSV and immunological immaturity may contribute to reduced immune responses and increased susceptibility to RSV disease and reinfection in young infants.

Genetic mechanisms of Coxiella burnetii lipopolysaccharide phase variation.

Coxiella burnetii is an intracellular pathogen that causes human Q fever, a disease that normally presents as a severe flu-like illness. Due to high infectivity and disease severity, the pathogen is considered a risk group 3 organism. Full-length lipopolysaccharide (LPS) is required for full virulence and disease by C. burnetii and is the only virulence factor currently defined by infection of an immunocompetent animal. Transition of virulent phase I bacteria with smooth LPS, to avirulent phase II bacteria with rough LPS, occurs during in vitro passage. Semi-rough intermediate forms are also observed. Here, the genetic basis of LPS phase conversion was investigated to obtain a more complete understanding of C. burnetii pathogenesis. Whole genome sequencing of strains producing intermediate and/or phase II LPS identified several common mutations in predicted LPS biosynthesis genes. After passage in broth culture for 30 weeks, phase I strains from different genomic groups exhibited similar phase transition kinetics and elevation of mutations in LPS biosynthesis genes. Targeted mutagenesis and genetic complementation using a new C. burnetii nutritional selection system based on lysine auxotrophy confirmed that six of the mutated genes were necessary for production of phase I LPS. Disruption of two of these genes in a C. burnetii phase I strain resulted in production of phase II LPS, suggesting inhibition of the encoded enzymes could represent a new therapeutic strategy for treatment of Q fever. Additionally, targeted mutagenesis of genes encoding LPS biosynthesis enzymes can now be used to construct new phase II strains from different genomic groups for use in pathogen-host studies at a risk group 2 level.

Type I interferon signaling attenuates regulatory T cell function in viral infection and in the tumor microenvironment.

Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute, chronic infectious diseases and tumor microenvironment remains unclear. We recently demonstrated that IFN-α/ß receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice in acute LCMV Armstrong, chronic Clone-13 viral infection, and in tumor models. In both viral infection and tumor models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. LCMV-specific CD8+ T cells and tumor infiltrating lymphocytes from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral and antitumor IFN-γ and TNF-α. In chronic viral model, the numbers of antiviral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and higher tumor burden than the WT controls. The enhanced activated phenotype was evident through transcriptome analysis of IFNARfl/flxFoxp3YFP-Cre mice Tregs during infection demonstrated differential expression of a unique gene signature characterized by elevated levels of genes involved in suppression and decreased levels of genes mediating apoptosis. Thus, IFN signaling in Tregs is beneficial to host resulting in a more effective antiviral response and augmented antitumor immunity.