Effects of N-glycan modifications on spike expression, virus infectivity, and neutralization sensitivity in ancestral compared to Omicron SARS-CoV-2 variants.

The SARS-CoV-2 spike glycoprotein has 22 potential N-linked glycosylation sites per monomer that are highly conserved among diverse variants, but how individual glycans affect virus entry and neutralization of Omicron variants has not been extensively characterized. Here we compared the effects of specific glycan deletions or modifications in the Omicron BA.1 and D614G spikes on spike expression, processing, and incorporation into pseudoviruses, as well as on virus infectivity and neutralization by therapeutic antibodies. We found that loss of potential glycans at spike residues N717 and N801 each conferred a loss of pseudovirus infectivity for Omicron but not for D614G or Delta variants. This decrease in infectivity correlated with decreased spike processing and incorporation into Omicron pseudoviruses. Oligomannose-enriched Omicron pseudoviruses generated in GnTI- cells or in the presence of kifunensine were non-infectious, whereas D614G or Delta pseudoviruses generated under similar conditions remained infectious. Similarly, growth of live (authentic) SARS-CoV-2 in the presence of kifunensine resulted in a greater reduction of titers for the BA.1.1 variant than Delta or D614G variants relative to their respective, untreated controls. Finally, we found that loss of some N-glycans, including N343 and N234, increased the maximum percent neutralization by the class 3 S309 monoclonal antibody against D614G but not BA.1 variants, while these glycan deletions altered the neutralization potency of the class 1 COV2-2196 and Etesevimab monoclonal antibodies without affecting maximum percent neutralization. The maximum neutralization by some antibodies also varied with the glycan composition, with oligomannose-enriched pseudoviruses conferring the highest percent neutralization. These results highlight differences in the interactions between glycans and residues among SARS-CoV-2 variants that can affect spike expression, virus infectivity, and susceptibility of variants to antibody neutralization.

Characterization of Entry Pathways, Species-Specific Angiotensin-Converting Enzyme 2 Residues Determining Entry, and Antibody Neutralization Evasion of Omicron BA.1, BA.1.1, BA.2, and BA.3 Variants.

The SARS-CoV-2 Omicron variants were first detected in November 2021, and several Omicron lineages (BA.1, BA.2, BA.3, BA.4, and BA.5) have since rapidly emerged. Studies characterizing the mechanisms of Omicron variant infection and sensitivity to neutralizing antibodies induced upon vaccination are ongoing by several groups. In the present study, we used pseudoviruses to show that the transmembrane serine protease 2 (TMPRSS2) enhances infection of BA.1, BA.1.1, BA.2, and BA.3 Omicron variants to a lesser extent than ancestral D614G. We further show that Omicron variants have higher sensitivity to inhibition by soluble angiotensin-converting enzyme 2 (ACE2) and the endosomal inhibitor chloroquine compared to D614G. The Omicron variants also more efficiently used ACE2 receptors from 9 out of 10 animal species tested, and unlike the D614G variant, used mouse ACE2 due to the Q493R and Q498R spike substitutions. Finally, neutralization of the Omicron variants by antibodies induced by three doses of Pfizer/BNT162b2 mRNA vaccine was 7- to 8-fold less potent than the D614G. These results provide insights into the transmissibility and immune evasion capacity of the emerging Omicron variants to curb their ongoing spread. IMPORTANCE The ongoing emergence of SARS-CoV-2 Omicron variants with an extensive number of spike mutations poses a significant public health and zoonotic concern due to enhanced transmission fitness and escape from neutralizing antibodies. We studied three Omicron lineage variants (BA.1, BA.2, and BA.3) and found that transmembrane serine protease 2 has less influence on Omicron entry into cells than on D614G, and Omicron exhibits greater sensitivity to endosomal entry inhibition compared to D614G. In addition, Omicron displays more efficient usage of diverse animal species ACE2 receptors than D614G. Furthermore, due to Q493R/Q498R substitutions in spike, Omicron, but not D614G, can use the mouse ACE2 receptor. Finally, three doses of Pfizer/BNT162b2 mRNA vaccination elicit high neutralization titers against Omicron variants, although the neutralization titers are still 7- to 8-fold lower those that against D614G. These results may give insights into the transmissibility and immune evasion capacity of the emerging Omicron variants to curb their ongoing spread.

The trispecific DARPin ensovibep inhibits diverse SARS-CoV-2 variants.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).

Measuring Neutralizing Antibodies to SARS-CoV-2 Using Lentiviral Spike-Pseudoviruses.

Assays measuring neutralizing antibodies (nAbs) against SARS-CoV-2 are used to evaluate serological responses after SARS-CoV-2 infection and the potency of therapeutic antibodies and preventive vaccines. It is therefore imperative that neutralization assays be sensitive, specific, quantitative, and scalable for high throughput. Pseudoviruses are excellent surrogates for highly pathogenic viruses such as SARS-CoV-2 because they can be safely used to measure nAbs in a biosafety level-2 laboratory. In addition, pseudoviruses allow for easy introduction of mutations to study the effect of amino acid changes in the spike protein. In this chapter, we describe a recently optimized assay for measuring neutralizing antibodies to SARS-CoV-2 that uses a HIV-based lentiviral vector pseudotyped with the spike glycoprotein of SARS-CoV-2 to infect 293T cells stably expressing ACE2 and TMPRSS2.

SARS-CoV-2 BA.1 variant is neutralized by vaccine booster-elicited serum but evades most convalescent serum and therapeutic antibodies.

The rapid spread of the highly contagious Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) along with its high number of mutations in the spike gene has raised alarms about the effectiveness of current medical countermeasures. To address this concern, we measured the neutralization of the Omicron BA.1 variant pseudovirus by postvaccination serum samples after two and three immunizations with the Pfizer/BioNTech162b2 SARS-CoV-2 mRNA (Pfizer/BNT162b2) vaccine, convalescent serum samples from unvaccinated individuals infected by different variants, and clinical-stage therapeutic antibodies. We found that titers against the Omicron variant were low or undetectable after two immunizations and in many convalescent serum samples, regardless of the infecting variant. A booster vaccination increased titers more than 30-fold against Omicron to values comparable to those seen against the D614G variant after two immunizations. Neither age nor sex was associated with the differences in postvaccination antibody responses. We also evaluated 18 clinical-stage therapeutic antibody products and an antibody mimetic protein product obtained directly from the manufacturers. Five monoclonal antibodies, the antibody mimetic protein, three antibody cocktails, and two polyclonal antibody preparations retained measurable neutralization activity against Omicron with a varying degree of potency. Of these, only three retained potencies comparable to the D614G variant. Two therapeutic antibody cocktails in the tested panel that are authorized for emergency use in the United States did not neutralize Omicron. These findings underscore the potential benefit of mRNA vaccine boosters for protection against Omicron and the need for rapid development of antibody therapeutics that maintain potency against emerging variants.

Key Substitutions in the Spike Protein of SARS-CoV-2 Variants Can Predict Resistance to Monoclonal Antibodies, but Other Substitutions Can Modify the Effects.

Mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can compromise the effectiveness of therapeutic antibodies. Most clinical-stage therapeutic antibodies target the spike receptor binding domain (RBD), but variants often have multiple mutations in several spike regions. To help predict antibody potency against emerging variants, we evaluated 25 clinical-stage therapeutic antibodies for neutralization activity against 60 pseudoviruses bearing spikes with single or multiple substitutions in several spike domains, including the full set of substitutions in B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.429 (epsilon), B.1.526 (iota), A.23.1, and R.1 variants. We found that 14 of 15 single antibodies were vulnerable to at least one RBD substitution, but most combination and polyclonal therapeutic antibodies remained potent. Key substitutions in variants with multiple spike substitutions predicted resistance, but the degree of resistance could be modified in unpredictable ways by other spike substitutions that may reside outside the RBD. These findings highlight the importance of assessing antibody potency in the context of all substitutions in a variant and show that epistatic interactions in spike can modify virus susceptibility to therapeutic antibodies. IMPORTANCE Therapeutic antibodies are effective in preventing severe disease from SARS-CoV-2 infection (COVID-19), but their effectiveness may be reduced by virus variants with mutations affecting the spike protein. To help predict resistance to therapeutic antibodies in emerging variants, we profiled resistance patterns of 25 antibody products in late stages of clinical development against a large panel of variants that include single and multiple substitutions found in the spike protein. We found that the presence of a key substitution in variants with multiple spike substitutions can predict resistance against a variant but that other substitutions can affect the degree of resistance in unpredictable ways. These findings highlight complex interactions among substitutions in the spike protein affecting virus neutralization and, potentially, virus entry into cells.

SARS-CoV-2 Delta Variant Displays Moderate Resistance to Neutralizing Antibodies and Spike Protein Properties of Higher Soluble ACE2 Sensitivity, Enhanced Cleavage and Fusogenic Activity.

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. Here, we compared B.1.617 variants for neutralization resistance by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. B.1.617.1, B.1.617.2, and AY.1 pseudoviruses showed a modest 1.5- to 4.4-fold reduction in neutralization by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for C.37, P.1, R.1, and B.1.526 pseudoviruses, but 7- and 16-fold reductions for vaccine-elicited and convalescent sera, respectively, were seen for B.1.351 pseudoviruses. Among twenty-three therapeutic antibodies tested, four antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses and six antibodies showed either complete or partial loss of neutralization against B.1.617.1 and AY.1 pseudoviruses. Our results indicate that the current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants. Finally, the P681R substitution confers efficient cleavage of B.1.617 variants' spike proteins and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.

SARS-COV-2 Delta variant displays moderate resistance to neutralizing antibodies and spike protein properties of higher soluble ACE2 sensitivity, enhanced cleavage and fusogenic activity.

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. The spike proteins of B.1.617.1, B.1.617.2, and AY.1 variants have several substitutions in the receptor binding domain (RBD), including L452R+E484Q, L452R+T478K, and K417N+L452R+T478K, respectively, that could potentially reduce effectiveness of therapeutic antibodies and current vaccines. Here we compared B.1.617 variants, and their single and double RBD substitutions for resistance to neutralization by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. Pseudoviruses with the B.1.617.1, B.1.617.2, and AY.1 spike showed a modest 1.5 to 4.4-fold reduction in neutralization titer by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for pseudoviruses with C.37, P.1, R.1, and B.1.526 spikes, but seven- and sixteen-fold reduction for vaccine-elicited and convalescent sera, respectively, was seen for pseudoviruses with the B.1.351 spike. Four of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses due to the L452R substitution, whereas six of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.1 pseudoviruses due to either the E484Q or L452R substitution. Against AY.1 pseudoviruses, the L452R and K417N substitutions accounted for the loss of neutralization by four antibodies and one antibody, respectively, whereas one antibody lost potency that could not be fully accounted for by a single RBD substitution. The modest resistance of B.1.617 variants to vaccine-elicited sera suggest that current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants, but the therapeutic antibodies need to be carefully selected based on their resistance profiles. Finally, the spike proteins of B.1.617 variants are more efficiently cleaved due to the P681R substitution, and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.

Interplay between RNA Viruses and Promyelocytic Leukemia Nuclear Bodies.

Promyelocytic leukemia nuclear bodies (PML NBs) are nuclear membrane-less sub structures that play a critical role in diverse cellular pathways including cell proliferation, DNA damage, apoptosis, transcriptional regulation, stem cell renewal, alternative lengthening of telomeres, chromatin organization, epigenetic regulation, protein turnover, autophagy, intrinsic and innate antiviral immunity. While intrinsic and innate immune functions of PML NBs or PML NB core proteins are well defined in the context of nuclear replicating DNA viruses, several studies also confirm their substantial roles in the context of RNA viruses. In the present review, antiviral activities of PML NBs or its core proteins on diverse RNA viruses that replicate in cytoplasm or the nucleus were discussed. In addition, viral counter mechanisms that reorganize PML NBs, and specifically how viruses usurp PML NB functions in order to create a cellular environment favorable for replication and pathogenesis, are also discussed.

Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2.

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

SARS-CoV-2 Omicron neutralization by therapeutic antibodies, convalescent sera, and post-mRNA vaccine booster.

The rapid spread of the highly contagious Omicron variant of SARS-CoV-2 along with its high number of mutations in the spike gene has raised alarm about the effectiveness of current medical countermeasures. To address this concern, we measured neutralizing antibodies against Omicron in three important settings: (1) post-vaccination sera after two and three immunizations with the Pfizer/BNT162b2 vaccine, (2) convalescent sera from unvaccinated individuals infected by different variants, and (3) clinical-stage therapeutic antibodies. Using a pseudovirus neutralization assay, we found that titers against Omicron were low or undetectable after two immunizations and in most convalescent sera. A booster vaccination significantly increased titers against Omicron to levels comparable to those seen against the ancestral (D614G) variant after two immunizations. Neither age nor sex were associated with differences in post-vaccination antibody responses. Only three of 24 therapeutic antibodies tested retained their full potency against Omicron and high-level resistance was seen against fifteen. These findings underscore the potential benefit of booster mRNA vaccines for protection against Omicron and the need for additional therapeutic antibodies that are more robust to highly mutated variants. ONE SENTENCE SUMMARY: Third dose of Pfizer/BioNTech COVID-19 vaccine significantly boosts neutralizing antibodies to the Omicron variant compared to a second dose, while neutralization of Omicron by convalescent sera, two-dose vaccine-elicited sera, or therapeutic antibodies is variable and often low.

Extracellular Vesicles from Red Blood Cells and Their Evolving Roles in Health, Coagulopathy and Therapy.

Red blood cells (RBCs) release extracellular vesicles (EVs) including both endosome-derived exosomes and plasma-membrane-derived microvesicles (MVs). RBC-derived EVs (RBCEVs) are secreted during erythropoiesis, physiological cellular aging, disease conditions, and in response to environmental stressors. RBCEVs are enriched in various bioactive molecules that facilitate cell to cell communication and can act as markers of disease. RBCEVs contribute towards physiological adaptive responses to hypoxia as well as pathophysiological progression of diabetes and genetic non-malignant hematologic disease. Moreover, a considerable number of studies focus on the role of EVs from stored RBCs and have evaluated post transfusion consequences associated with their exposure. Interestingly, RBCEVs are important contributors toward coagulopathy in hematological disorders, thus representing a unique evolving area of study that can provide insights into molecular mechanisms that contribute toward dysregulated hemostasis associated with several disease conditions. Relevant work to this point provides a foundation on which to build further studies focused on unraveling the potential roles of RBCEVs in health and disease. In this review, we provide an analysis and summary of RBCEVs biogenesis, composition, and their biological function with a special emphasis on RBCEV pathophysiological contribution to coagulopathy. Further, we consider potential therapeutic applications of RBCEVs.

A comparison of exosome purification methods using serum of Marek's disease virus (MDV)-vaccinated and -tumor-bearing chickens.

Marek's disease (MD) is an alphaherpesvirus (Marek's disease virus, MDV)-induced pathology of chickens associated with paralysis, immunosuppression, neurological signs, and T-cell lymphomas. MD is controlled in poultry production via live attenuated vaccines. The purpose of the current study was to compare methods for precipitating exosomes from vaccinated and protected chicken sera (VEX) and tumor-bearing chicken sera (TEX) for biomarker analysis of vaccine-induced protection and MD lymphomas respectively. A standard polyethylene glycol (PEG, 8%) method was compared to a commercial reagent (total exosome isolation reagent, TEI) for exosome yield and RNA content. Although exosomes purified by PEG or TEI were comparable in size and morphology, TEI-reagent yielded 3-4-fold greater concentration. Relative expression of 8 out of 10 G. gallus- and MDV1-encoded miRNAs examined displayed significant difference depending upon the precipitation method used. Standard PEG yields comparable, albeit lower amounts of exosomes than the TEI-reagent and a distinctive miRNA composition.

A Review on SARS-CoV-2 Virology, Pathophysiology, Animal Models, and Anti-Viral Interventions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of CoV disease 2019 (COVID-19) is a highly pathogenic and transmissible CoV that is presently plaguing the global human population and economy. No proven effective antiviral therapy or vaccine currently exists, and supportive care remains to be the cornerstone treatment. Through previous lessons learned from SARS-CoV-1 and MERS-CoV studies, scientific groups worldwide have rapidly expanded the knowledge pertaining to SARS-CoV-2 virology that includes in vitro and in vivo models for testing of antiviral therapies and randomized clinical trials. In the present narrative, we review SARS-CoV-2 virology, clinical features, pathophysiology, and animal models with a specific focus on the antiviral and adjunctive therapies currently being tested or that require testing in animal models and randomized clinical trials.

Avian Pattern Recognition Receptor Sensing and Signaling.

Pattern recognition receptors (PRRs) are a class of immune sensors that play a critical role in detecting and responding to several conserved patterns of microorganisms. As such, they play a major role in the maintenance of immune homeostasis and anti-microbial defense. Fundamental knowledge pertaining to the discovery of PRR functions and their ligands continue to advance the understanding of immune system and disease resistance, which led to the rational design and/or application of various PRR ligands as vaccine adjuvants. In addition, the conserved nature of many PRRs throughout the animal kingdom has enabled the utilization of the comparative genomics approach in PRR identification and the study of evolution, structural features, and functions in many animal species including avian. In the present review, we focused on PRR sensing and signaling functions in the avian species, domestic chicken, mallard, and domestic goose. In addition to summarizing recent advances in the understanding of avian PRR functions, the present review utilized a comparative biology approach to identify additional PRRs, whose functions have been well studied in mammalians but await functional characterization in avian.

Transcriptional Analyses of Innate and Acquired Immune Patterning Elicited by Marek's Disease Virus Vaccine Strains: Turkey Herpesvirus (HVT), Marek's Disease Virus 2 (strain SB1), and Bivalent Vaccines (HVT/SB1 and HVT-LT/SB1).

Marek's disease (MD) is a complex pathology of chickens caused by MD virus (MDV) 1 and is observed as paralysis, immune suppression, neurologic signs, and the rapid formation of T-cell lymphomas. The incidence of MD in commercial broilers is largely controlled via vaccination, either in ovo or at hatch with live attenuated vaccines, i.e., turkey herpesvirus (HVT) or a bivalent combination of HVT with the MDV 2 strain (SB1). To further extend the protection conferred by bivalent HVT/SB-1, recombinant HVTs encoding transgenes of other avian viruses have similarly been used for in ovo administration. Despite decades of use, the specific mechanisms associated with vaccine-induced protection remain obscure. Additionally, the mechanistic basis for vaccine synergism conferred by bivalent HVT/SB-1, compared with HVT or SB-1 administered alone, is largely unknown. In the present study, we report on temporal changes in innate and acquired immune-patterning gene expression by using ex vivo splenocyte infection and in ovo vaccination models. We report that in the ex vivo splenocyte infection model, by 72 hr postinfection, vaccines induced IFN and IFN-stimulated gene expression, with lesser proinflammatory cytokine induction. For several genes (TLR3, IFN-γ, OASL, Mx1, NOS2A, and IL-1ß), the effects on gene expression were additive for HVT, SB1, and HVT/SB1 infection. We observed similar patterns of induction in in ovo-vaccinated commercial broiler embryos and chicks with HVT/SB-1 or recombinant HVT-based bivalent combination (HVT-LT/SB-1). Furthermore, HVT/SB-1 or HVT-LT/SB-1 in ovo vaccination appeared to hasten immune maturation, with expression patterns suggesting accelerated migration of T and natural killer cells into the spleen. Finally, HVT/SB-1 vaccination resulted in a coordinated induction of IL-12p40 and downregulation of suppressors of cytokine signaling 1 and 3, indicative of classical macrophage 1 and T-helper 1 patterning.

Análisis transcripcionales de patrones inmunes innatos y adquiridos inducidos por cepas vacunales del virus de la enfermedad de Marek: virus herpes del pavo (HVT), virus de Marek 2 (cepa SB1) y vacunas bivalentes (HVT/SB1 y HVT-LT/SB1). La enfermedad de Marek (MD) es una patología compleja de los pollos causada por el virus de Marek (MDV) 1 y se observa como parálisis, depresión inmune, signos neurológicos y la formación rápida de linfomas de células T. La incidencia de la enfermedad de Marek en pollos de engorde comerciales se controla en gran medida a través de la vacunación, ya sea in ovo o al momento de la eclosión con vacunas vivas atenuadas, por ejemplo, herpesvirus de pavo (HVT) o una combinación bivalente de HVT con la cepa SB1. Para ampliar aún más la protección conferida por la vacuna bivalente HVT/SB-1, los HVT recombinantes que codifican transgenes de otros virus aviares se han utilizado de forma similar para la administración in ovo. A pesar de décadas de uso, los mecanismos específicos asociados con la protección inducida por la vacuna siguen sin ser esclarecidos completamente. Además, el mecanismo para la sinergia de la vacuna conferida por la vacuna bivalente HVT/SB-1, en comparación con la administración de la cepa HVT o de la cepa SB-1 por sí solas, es en gran medida desconocida. En el presente estudio, se informa sobre los cambios temporales en la expresión genética de patrones inmunes innatos y adquiridos mediante la infección de esplenocitos ex vivo y en modelos de vacunación in ovo. Se reporta que en el modelo de infección de esplenocitos ex vivo, por 72 horas después de la infección, las vacunas indujeron IFN y la expresión de genes estimulada por IFN, con menor inducción de citocinas proinflamatorias. Para varios genes (TLR3, IFNc, OASL, Mx1, NOS2A e IL-1ß), los efectos sobre la expresión de genes fueron aditivos para la infección por HVT, SB1 y HVT/SB1. Se Observaron patrones de inducción similares en embriones de pollo y pollos de engorde comerciales vacunados in ovo con HVT/SB-1 o con la combinación bivalente recombinante basada en HVT (HVT-LT/SB-1). Además, la vacunación in ovo con HVT/SB-1 o HVT-LT/SB-1 parecen acelerar la maduración inmune, con patrones de expresión que sugieren una migración acelerada de células T y células asesinas naturales en el bazo. Finalmente, la vacuna HVT/SB-1 dio como resultado una inducción coordinada de IL-12p40 y una regulación a la baja de supresores de las señales de citocinas 1 y 3, indicativas de los patrones clásicos de macrófagos 1 y células cooperadoras tipo 1.

Comparison of the Transcriptomes and Proteomes of Serum Exosomes from Marek's Disease Virus-Vaccinated and Protected and Lymphoma-Bearing Chickens.

Marek's disease virus (MDV) is the causative agent of Marek's disease (MD), a complex pathology of chickens characterized by paralysis, immunosuppression, and T-cell lymphomagenesis. MD is controlled in poultry production via vaccines administered in ovo or at hatch, and these confer protection against lymphoma formation, but not superinfection by MDV field strains. Despite vaccine-induced humoral and cell-mediated immune responses, mechanisms eliciting systemic protection remain unclear. Here we report the contents of serum exosomes to assess their possible roles as indicators of systemic immunity, and alternatively, tumor formation. We examined the RNA and protein content of serum exosomes from CVI988 (Rispens)-vaccinated and protected chickens (VEX), and unvaccinated tumor-bearing chickens (TEX), via deep-sequencing and mass spectrometry, respectively. Bioinformatic analyses of microRNAs (miRNAs) and predicted miRNA targets indicated a greater abundance of tumor suppressor miRNAs in VEX compared to TEX. Conversely, oncomiRs originating from cellular (miRs 106a-363) and MDV miRNA clusters were more abundant in TEX compared to VEX. Most notably, mRNAs mapping to the entire MDV genome were identified in VEX, while mRNAs mapping to the repeats flanking the unique long (IRL/TRL) were identified in TEX. These data suggest that long-term systemic vaccine-induced immune responses may be mediated at the level of VEX which transfer viral mRNAs to antigen presenting cells systemically. Proteomic analyses of these exosomes suggested potential biomarkers for VEX and TEX. These data provide important putative insight into MDV-mediated immune suppression and vaccine responses, as well as potential serum biomarkers for MD protection and susceptibility.

Induction of the unfolded protein response (UPR) during Marek's disease virus (MDV) infection.

Marek's disease (MD) is a pathology of chickens associated with paralysis, immune suppression, and the rapid formation of T-cell lymphomas. MD is caused by the herpesvirus, Marek's disease virus (MDV). We examined endoplasmic reticulum (ER) stress and the activation of unfolded protein response (UPR) pathways during MDV infection of cells in culture and lymphocytes in vivo. MDV strains activate the UPR as measured by increased mRNA expression of GRP78/BiP with concomitant XBP1 splicing and induction of its target gene, EDEM1. Cell culture replication of virulent, but not vaccine MDVs, activated the UPR at late in infection. Pathotype-associated UPR activation was induced to a greater level by a vv + MDV. Discrete UPR activation was observed during MDV in vivo infection, with the level of UPR modulation being affected by the MDV oncoprotein Meq. Finally, ATF6 was found to be activated in vv + MDV-induced primary lymphomas, suggesting a possible role in tumor progression.

Evaluation and validation of reference gene stability during Marek's disease virus (MDV) infection.

Quantitative RT-PCR (qRT-PCR) is widely used in the study of relative gene expression in general, and has been used in the field of Marek's disease (MD) research to measure transcriptional responses to infection and/or vaccination. Studies in the past have either employed cellular ß-actin (BACT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference genes, although the stability of their expression in the context of Marek's disease virus (MDV) infection has never been investigated. In the present study, we compared the stability of five reference genes (BACT, 28S RNA, 18S RNA, GAPDH, Peptidyl-prolyl-isomerase B [PPIB], a.k.a. cyclophilin B) as standard internal controls in chicken embryo fibroblast (CEFs) cultures infected with either MD vaccine or oncogenic MDV1 viruses. We further extend these analyses to reference gene stability in spleen lymphomas induced by infection of commercial broiler chickens with a very virulent plus MDV1 (vv+ TK-2a virus). Two excel based algorithms, (Bestkeeper and Normfinder) were employed to compare reference gene stability. Bestkeeper and Normfinder analysis of reference gene stability in virus- and mock-infected cells, showed that 28S RNA and PPIB displayed higher stability in CEF infections with either oncogenic or vaccine viruses. In addition, both Bestkeeper and Normfinder determined 28S RNA and PPIB to be the most stably-expressed reference genes in vivo in vv+ TK-2a-induced spleen lymphomas. Furthermore, Bestkeeper and Normfinder analyses both determined BACT to be the least stable reference gene during MDV infection of CEF with oncogenic viruses, vaccine viruses, as well as in vv+ TK-2a-induced spleen lymphomas.