Structural and functional characterizations of infectivity and immune evasion of SARS-CoV-2 Omicron.

The SARS-CoV-2 Omicron variant with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the spike (S) from Omicron reveals amino acid substitutions forging interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of the viral fusion step. Alterations in local conformation, charge, and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Structure of the Omicron S bound with human ACE2, together with the analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members, as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies, sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

ICP22/IE63 Mediated Transcriptional Regulation and Immune Evasion: Two Important Survival Strategies for Alphaherpesviruses.

In the process of infecting the host, alphaherpesviruses have derived a series of adaptation and survival strategies, such as latent infection, autophagy and immune evasion, to survive in the host environment. Infected cell protein 22 (ICP22) or its homologue immediate early protein 63 (IE63) is a posttranslationally modified multifunctional viral regulatory protein encoded by all alphaherpesviruses. In addition to playing an important role in the efficient use of host cell RNA polymerase II, it also plays an important role in the defense process of the virus overcoming the host immune system. These two effects of ICP22/IE63 are important survival strategies for alphaherpesviruses. In this review, we summarize the complex mechanism by which the ICP22 protein regulates the transcription of alphaherpesviruses and their host genes and the mechanism by which ICP22/IE63 participates in immune escape. Reviewing these mechanisms will also help us understand the pathogenesis of alphaherpesvirus infections and provide new strategies to combat these viral infections.

B.1.617.2 enters and fuses lung cells with increased efficiency and evades antibodies induced by infection and vaccination.

The Delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), B.1.617.2, emerged in India and has spread to over 80 countries. B.1.617.2 replaced B.1.1.7 as the dominant virus in the United Kingdom, resulting in a steep increase in new infections, and a similar development is expected for other countries. Effective countermeasures require information on susceptibility of B.1.617.2 to control by antibodies elicited by vaccines and used for coronavirus disease 2019 (COVID-19) therapy. We show, using pseudotyping, that B.1.617.2 evades control by antibodies induced upon infection and BNT162b2 vaccination, although to a lesser extent as compared to B.1.351. We find that B.1.617.2 is resistant against bamlanivimab, a monoclonal antibody with emergency use authorization for COVID-19 therapy. Finally, we show increased Calu-3 lung cell entry and enhanced cell-to-cell fusion of B.1.617.2, which may contribute to augmented transmissibility and pathogenicity of this variant. These results identify B.1.617.2 as an immune evasion variant with increased capacity to enter and fuse lung cells.

HIV-1 Establishes a Sanctuary Site in the Testis by Permeating the BTB Through Changes in Cytoskeletal Organization.

Studies suggest that HIV-1 invades the testis through initial permeation of the blood-testis barrier (BTB). The selectivity of the BTB to antiretroviral drugs makes this site a sanctuary for the virus. Little is known about how HIV-1 crosses the BTB and invades the testis. Herein, we used 2 approaches to examine the underlying mechanism(s) by which HIV-1 permeates the BTB and gains entry into the seminiferous epithelium. First, we examined if recombinant Tat protein was capable of perturbing the BTB and making the barrier leaky, using the primary rat Sertoli cell in vitro model that mimics the BTB in vivo. Second, we used HIV-1-infected Sup-T1 cells to investigate the activity of HIV-1 infection on cocultured Sertoli cells. Using both approaches, we found that the Sertoli cell tight junction permeability barrier was considerably perturbed and that HIV-1 effectively permeates the BTB by inducing actin-, microtubule-, vimentin-, and septin-based cytoskeletal changes in Sertoli cells. These studies suggest that HIV-1 directly perturbs BTB function, potentially through the activity of the Tat protein.

Definition of the immune evasion-replication interface of rabies virus P protein.

Rabies virus phosphoprotein (P protein) is a multifunctional protein that plays key roles in replication as the polymerase cofactor that binds to the complex of viral genomic RNA and the nucleoprotein (N protein), and in evading the innate immune response by binding to STAT transcription factors. These interactions are mediated by the C-terminal domain of P (PCTD). The colocation of these binding sites in the small globular PCTD raises the question of how these interactions underlying replication and immune evasion, central to viral infection, are coordinated and, potentially, coregulated. While direct data on the binding interface of the PCTD for STAT1 is available, the lack of direct structural data on the sites that bind N protein limits our understanding of this interaction hub. The PCTD was proposed to bind via two sites to a flexible loop of N protein (Npep) that is not visible in crystal structures, but no direct analysis of this interaction has been reported. Here we use Nuclear Magnetic Resonance, and molecular modelling to show N protein residues, Leu381, Asp383, Asp384 and phosphor-Ser389, are likely to bind to a 'positive patch' of the PCTD formed by Lys211, Lys214 and Arg260. Furthermore, in contrast to previous predictions we identify a single site of interaction on the PCTD by this Npep. Intriguingly, this site is proximal to the defined STAT1 binding site that includes Ile201 to Phe209. However, cell-based assays indicate that STAT1 and N protein do not compete for P protein. Thus, it appears that interactions critical to replication and immune evasion can occur simultaneously with the same molecules of P protein so that the binding of P protein to activated STAT1 can potentially occur without interrupting interactions involved in replication. These data suggest that replication complexes might be directly involved in STAT1 antagonism.

PlzA is a bifunctional c-di-GMP biosensor that promotes tick and mammalian host-adaptation of Borrelia burgdorferi.

In this study, we examined the relationship between c-di-GMP and its only known effector protein, PlzA, in Borrelia burgdorferi during the arthropod and mammalian phases of the enzootic cycle. Using a B. burgdorferi strain expressing a plzA point mutant (plzA-R145D) unable to bind c-di-GMP, we confirmed that the protective function of PlzA in ticks is c-di-GMP-dependent. Unlike ΔplzA spirochetes, which are severely attenuated in mice, the plzA-R145D strain was fully infectious, firmly establishing that PlzA serves a c-di-GMP-independent function in mammals. Contrary to prior reports, loss of PlzA did not affect expression of RpoS or RpoS-dependent genes, which are essential for transmission, mammalian host-adaptation and murine infection. To ascertain the nature of PlzA's c-di-GMP-independent function(s), we employed infection models using (i) host-adapted mutant spirochetes for needle inoculation of immunocompetent mice and (ii) infection of scid mice with in vitro-grown organisms. Both approaches substantially restored ΔplzA infectivity, suggesting that PlzA enables B. burgdorferi to overcome an early bottleneck to infection. Furthermore, using a Borrelia strain expressing a heterologous, constitutively active diguanylate cyclase, we demonstrate that 'ectopic' production of c-di-GMP in mammals abrogates spirochete virulence and interferes with RpoS function at the post-translational level in a PlzA-dependent manner. Structural modeling and SAXS analysis of liganded- and unliganded-PlzA revealed marked conformational changes that underlie its biphasic functionality. This structural plasticity likely enables PlzA to serve as a c-di-GMP biosensor that in its respective liganded and unliganded states promote vector- and host-adaptation by the Lyme disease spirochete.

Host immunity increases Mycobacterium tuberculosis reliance on cytochrome bd oxidase.

In order to sustain a persistent infection, Mycobacterium tuberculosis (Mtb) must adapt to a changing environment that is shaped by the developing immune response. This necessity to adapt is evident in the flexibility of many aspects of Mtb metabolism, including a respiratory chain that consists of two distinct terminal cytochrome oxidase complexes. Under the conditions tested thus far, the bc1/aa3 complex appears to play a dominant role, while the alternative bd oxidase is largely redundant. However, the presence of two terminal oxidases in this obligate pathogen implies that respiratory requirements might change during infection. We report that the cytochrome bd oxidase is specifically required for resisting the adaptive immune response. While the bd oxidase was dispensable for growth in resting macrophages and the establishment of infection in mice, this complex was necessary for optimal fitness after the initiation of adaptive immunity. This requirement was dependent on lymphocyte-derived interferon gamma (IFNγ), but did not involve nitrogen and oxygen radicals that are known to inhibit respiration in other contexts. Instead, we found that ΔcydA mutants were hypersusceptible to the low pH encountered in IFNγ-activated macrophages. Unlike wild type Mtb, cytochrome bd-deficient bacteria were unable to sustain a maximal oxygen consumption rate (OCR) at low pH, indicating that the remaining cytochrome bc1/aa3 complex is preferentially inhibited under acidic conditions. Consistent with this model, the potency of the cytochrome bc1/aa3 inhibitor, Q203, is dramatically enhanced at low pH. This work identifies a critical interaction between host immunity and pathogen respiration that influences both the progression of the infection and the efficacy of potential new TB drugs.

Host tropism determination by convergent evolution of immunological evasion in the Lyme disease system.

Pathogens possess the ability to adapt and survive in some host species but not in others-an ecological trait known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. Three main causative agents of LD, Borrelia burgdorferi, B. afzelii, and B. garinii, vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different Borrelia species, utilizing feeding chambers and live mice and quail, we found species-level differences in bacterial transmission. These differences localize on the tick blood meal, and specifically complement, a defense in vertebrate blood, and a polymorphic bacterial protein, CspA, which inactivates complement by binding to a host complement inhibitor, Factor H (FH). CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. CspA is the only member of the Pfam54 gene family to exhibit host-specific FH-binding. Phylogenetic analyses revealed convergent evolution as the driver of such uniqueness, and that FH-binding likely emerged during the last glacial maximum. Our results identify a determinant of host tropism in Lyme disease infection, thus defining an evolutionary mechanism that shapes host-pathogen associations.

In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus.

Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.

Hepatitis C virus modulates signal peptide peptidase to alter host protein processing.

Immunoevasins are viral proteins that prevent antigen presentation on major histocompatibility complex (MHC) class I, thus evading host immune recognition. Hepatitis C virus (HCV) evades immune surveillance to induce chronic infection; however, how HCV-infected hepatocytes affect immune cells and evade immune recognition remains unclear. Herein, we demonstrate that HCV core protein functions as an immunoevasin. Its expression interfered with the maturation of MHC class I molecules catalyzed by the signal peptide peptidase (SPP) and induced their degradation via HMG-CoA reductase degradation 1 homolog, thereby impairing antigen presentation to CD8+ T cells. The expression of MHC class I in the livers of HCV core transgenic mice and chronic hepatitis C patients was impaired but was restored in patients achieving sustained virological response. Finally, we show that the human cytomegalovirus US2 protein, possessing a transmembrane region structurally similar to the HCV core protein, targets SPP to impair MHC class I molecule expression. Thus, SPP represents a potential target for the impairment of MHC class I molecules by DNA and RNA viruses.

Overexpression of S100A9 in tumor stroma contribute to immune evasion of NK/T cell lymphoma and predict poor response rate.

NK/T cell lymphoma (NKTCL) represents an aggressive lymphoid malignancy characterized by dismal prognosis. Immune-checkpoint blockade has shown promising efficacy in NKTCL. However, the molecular mechanisms underlying immune evasion in NKTCL have never been explored. Here, proteomic analysis was used to identify the differentially expressed proteins between NKTCL patients and healthy individuals. We found that S100A9, an immunosuppressive molecule, was much higher in NKTCL patients both in serum and tumor stroma. Elevated level of S100A9 was associated with advanced stage, poor overall response and early recurrence. Moreover, percentage of myeloid-derived suppressor cells (MDSCs) in peripheral blood was positively correlated with levels of S100A9. Low concentration of S100A9 promoted proliferation of NKTCL cells, while did not affect cell apoptosis and cell cycles. Furthermore, programmed death ligand 1 (PD-L1) expression on NKTCL cells was up-regulated by S100A9 through activation of ERK1/2 signaling. Inhibition of ERK1/2 signaling significantly decreased tumor growth and PD-L1 expression induced by S100A9. In conclusion, our research firstly identified S100A9 as an immune suppressor in the tumorigenesis of NKTCL via accumulation of MDSCs and upregulation of PD-L1 expression. S100A9 may serve as a potential target to increase the efficacy of immunotherapy in NKTCL.

Comparative assessment of immune evasion mechanisms in human whole-blood infection assays by a systems biology approach.

Computer simulations of mathematical models open up the possibility of assessing hypotheses generated by experiments on pathogen immune evasion in human whole-blood infection assays. We apply an interdisciplinary systems biology approach in which virtual infection models implemented for the dissection of specific immune mechanisms are combined with experimental studies to validate or falsify the respective hypotheses. Focusing on the assessment of mechanisms that enable pathogens to evade the immune response in the early time course of a whole-blood infection, the least-square error (LSE) as a measure for the quantitative agreement between the theoretical and experimental kinetics is combined with the Akaike information criterion (AIC) as a measure for the model quality depending on its complexity. In particular, we compare mathematical models with three different types of pathogen immune evasion as well as all their combinations: (i) spontaneous immune evasion, (ii) evasion mediated by immune cells, and (iii) pre-existence of an immune-evasive pathogen subpopulation. For example, by testing theoretical predictions in subsequent imaging experiments, we demonstrate that the simple hypothesis of having a subpopulation of pre-existing immune-evasive pathogens can be ruled out. Furthermore, in this study we extend our previous whole-blood infection assays for the two fungal pathogens Candida albicans and C. glabrata by the bacterial pathogen Staphylococcus aureus and calibrated the model predictions to the time-resolved experimental data for each pathogen. Our quantitative assessment generally reveals that models with a lower number of parameters are not only scored with better AIC values, but also exhibit lower values for the LSE. Furthermore, we describe in detail model-specific and pathogen-specific patterns in the kinetics of cell populations that may be measured in future experiments to distinguish and pinpoint the underlying immune mechanisms.

TcpC inhibits toll-like receptor signaling pathway by serving as an E3 ubiquitin ligase that promotes degradation of myeloid differentiation factor 88.

TcpC is a virulence factor of uropathogenic E. coli (UPEC). It was found that TIR domain of TcpC impedes TLR signaling by direct association with MyD88. It has been a long-standing question whether bacterial pathogens have evolved a mechanism to manipulate MyD88 degradation by ubiquitin-proteasome pathway. Here, we show that TcpC is a MyD88-targeted E3 ubiquitin ligase. Kidney macrophages from mice with pyelonephritis induced by TcpC-secreting UPEC showed significantly decreased MyD88 protein levels. Recombinant TcpC (rTcpC) dose-dependently inhibited protein but not mRNA levels of MyD88 in macrophages. Moreover, rTcpC significantly promoted MyD88 ubiquitination and accumulation in proteasomes in macrophages. Cys12 and Trp106 in TcpC are crucial amino acids in maintaining its E3 activity. Therefore, TcpC blocks TLR signaling pathway by degradation of MyD88 through ubiquitin-proteasome system. Our findings provide not only a novel biochemical mechanism underlying TcpC-medicated immune evasion, but also the first example that bacterial pathogens inhibit MyD88-mediated signaling pathway by virulence factors that function as E3 ubiquitin ligase.

Insight into the emerging role of SARS-CoV-2 nonstructural and accessory proteins in modulation of multiple mechanisms of host innate defense.

Coronavirus disease-19 (COVID-19) is an extremely infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has become a major global health concern. The induction of a coordinated immune response is crucial to the elimination of any pathogenic infection. However, SARS-CoV-2 can modulate the host immune system to favor viral adaptation and persistence within the host. The virus can counteract type I interferon (IFN-I) production, attenuating IFN-I signaling pathway activation and disrupting antigen presentation. Simultaneously, SARS-CoV-2 infection can enhance apoptosis and the production of inflammatory mediators, which ultimately results in increased disease severity. SARS-CoV-2 produces an array of effector molecules, including nonstructural proteins (NSPs) and open-reading frames (ORFs) accessory proteins. We describe the complex molecular interplay of SARS-CoV-2 NSPs and accessory proteins with the host's signaling mediating immune evasion in the current review. In addition, the crucial role played by immunomodulation therapy to address immune evasion is discussed. Thus, the current review can provide new directions for the development of vaccines and specific therapies.

KRAS drives immune evasion in a genetic model of pancreatic cancer.

Immune evasion is a hallmark of KRAS-driven cancers, but the underlying causes remain unresolved. Here, we use a mouse model of pancreatic ductal adenocarcinoma to inactivate KRAS by CRISPR-mediated genome editing. We demonstrate that at an advanced tumor stage, dependence on KRAS for tumor growth is reduced and is manifested in the suppression of antitumor immunity. KRAS-deficient cells retain the ability to form tumors in immunodeficient mice. However, they fail to evade the host immune system in syngeneic wild-type mice, triggering strong antitumor response. We uncover changes both in tumor cells and host immune cells attributable to oncogenic KRAS expression. We identify BRAF and MYC as key mediators of KRAS-driven tumor immune suppression and show that loss of BRAF effectively blocks tumor growth in mice. Applying our results to human PDAC we show that lowering KRAS activity is likewise associated with a more vigorous immune environment.

Insect Immune Evasion by Dauer and Nondauer Entomopathogenic Nematodes.

The immune response of animals, including insects, is overcome by some parasites. For example, dauer larvae (DL) of the obligate entomopathogenic nematodes (EPNs) Heterorhabditis and Steinernema can invade insects, evade their defenses, and cause death. Although DL were long assumed to be the only infective stage of nematodes, recent reports suggest that L2-L3 larvae of facultative EPNs are also capable of killing insects. There are no studies, to our knowledge, about the role of nonimmunological barriers (the exoskeleton and its openings) in avoiding infection by DL and L2-L3 larvae, or whether these larval stages evade the host immune system in the same way. The objective of this study was to examine these questions by infecting Galleria mellonella with the facultative parasitic nematode Rhabditis regina. DL or L2-L3 larvae were either deposited on or near the moths or injected into their hemocoel. Once nematodes reached the hemocoel, the following host immune response parameters were quantified: prophenoloxidase, phenoloxidase, lytic activity, and the number of granular hemocytes. DL showed a greater ability to penetrate the exoskeleton than L2-L3 larvae. Once inside, however, both went unnoticed by the immune system and killed the insect. A higher number of granular hemocytes was activated by L2-L3 larvae than DL. We show for the first time that L2-L3 larvae can penetrate and evade the insect immune system. Further research is needed to compare facultative and specialized EPNs to determine which is more likely, with both DL and L2-L3 larvae, to evade insect defense barriers and produce death. The results will contribute to understanding the evolution of virulence in entomopathogenic nematodes.

Leishmania donovani infection suppresses Allograft Inflammatory Factor-1 in monocytes and macrophages to inhibit inflammatory responses.

Macrophages and monocytes are important for clearance of Leishmania infections. However, immune evasion tactics employed by the parasite results in suppressed inflammatory responses, marked by deficient macrophage functions and increased accumulation of monocytes. This results in an ineffective ability to clear parasite loads. Allograft Inflammatory Factor-1 (AIF1) is expressed in myeloid cells and serves to promote immune responses. However, AIF1 involvement in monocyte and macrophage functions during parasitic infections has not been explored. This study now shows that Leishmania donovani inhibits AIF1 expression in macrophages to block pro-inflammatory responses. Mice challenged with the parasite had markedly reduced AIF1 expression in splenic macrophages. Follow-up studies using in vitro approaches confirmed that L. donovani infection in macrophages suppresses AIF1 expression, which correlated with reduction in pro-inflammatory cytokine production and increased parasite load. Ectopic overexpression of AIF1 in macrophages provided protection from infection, marked by robust pro-inflammatory cytokine production and efficient pathogen clearance. Further investigations found that inhibiting AIF1 expression in bone marrow cells or monocytes impaired differentiation into functional macrophages. Collectively, results show that AIF1 is a critical regulatory component governing monocyte and macrophage immune functions and that L. donovani infection can suppress the gene as an immune evasion tactic.

The immune response and immune evasion characteristics in SARS-CoV, MERS-CoV, and SARS-CoV-2: Vaccine design strategies.

The worldwide outbreak of SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 as a novel human coronavirus, was the worrying news at the beginning of 2020. Since its emergence complicated more than 870,000 individuals and led to more than 43,000 deaths worldwide. Considering to the potential threat of a pandemic and transmission severity of it, there is an urgent need to evaluate and realize this new virus's structure and behavior and the immunopathology of this disease to find potential therapeutic protocols and to design and develop effective vaccines. This disease is able to agitate the response of the immune system in the infected patients, so ARDS, as a common consequence of immunopathological events for infections with Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV, and SARS-CoV-2, could be the main reason for death. Here, we summarized the immune response and immune evasion characteristics in SARS-CoV, MERS-CoV, and SARS-CoV-2 and therapeutic and prophylactic strategies with a focus on vaccine development and its challenges.

HIV-1 Vpr antagonizes innate immune activation by targeting karyopherin-mediated NF-κB/IRF3 nuclear transport.

HIV-1 must replicate in cells that are equipped to defend themselves from infection through intracellular innate immune systems. HIV-1 evades innate immune sensing through encapsidated DNA synthesis and encodes accessory genes that antagonize specific antiviral effectors. Here, we show that both particle associated, and expressed HIV-1 Vpr, antagonize the stimulatory effect of a variety of pathogen associated molecular patterns by inhibiting IRF3 and NF-κB nuclear transport. Phosphorylation of IRF3 at S396, but not S386, was also inhibited. We propose that, rather than promoting HIV-1 nuclear import, Vpr interacts with karyopherins to disturb their import of IRF3 and NF-κB to promote replication in macrophages. Concordantly, we demonstrate Vpr-dependent rescue of HIV-1 replication in human macrophages from inhibition by cGAMP, the product of activated cGAS. We propose a model that unifies Vpr manipulation of nuclear import and inhibition of innate immune activation to promote HIV-1 replication and transmission.