Production of polyclonal viperin antisera using N-terminal deleted recombinant bovine viperin.

Viperin, also known as radical S-adenosyl methionine domain-containing protein (RSAD2) is a multifunctional interferon-stimulated gene (ISG) that is activated during the viral infections. Viperin belongs to S-adenosyl methionine (SAM) superfamily of enzymes known to catalyze radical-mediated reactions and viperin inhibits a wide range of DNA and RNA viruses through its broad range of activity. The present study reports cloning and expression of bovine viperin in a bacterial expression system. PCR-based site-directed mutagenesis was carried out for deletion of N-terminal 1-70 amino acid containing amphipathic helix of viperin that interferes in protein expression and purification. The resultant truncated viperin protein was expressed in Escherichia coli, BL-21(DE3) competent cells and purified using nickel charged affinity column. The truncated 54 kDa protein was confirmed by western blot using human RSAD2 as a probe. Further, in house, hyperimmune serum was raised against the truncated viperin in the rabbit and the reactivity was confirmed by western blot using mammalian expression vector construct of viperin transfected in Baby Hamster kidney (BHK) cells and in MDBK cells infected with Foot and Mouth disease Asia I virus.

Immune serum-activated human macrophages coordinate with eosinophils to immobilize Ascaris suum larvae.

Helminth infection represents a major health problem causing approximately 5 million disability-adjusted life years worldwide. Concerns that repeated anti-helminthic treatment may lead to drug resistance render it important that vaccines are developed but will require increased understanding of the immune-mediated cellular and antibody responses to helminth infection. IL-4 or antibody-activated murine macrophages are known to immobilize parasitic nematode larvae, but few studies have addressed whether this is translatable to human macrophages. In the current study, we investigated the capacity of human macrophages to recognize and attack larval stages of Ascaris suum, a natural porcine parasite that is genetically similar to the human helminth Ascaris lumbricoides. Human macrophages were able to adhere to and trap A suum larvae in the presence of either human or pig serum containing Ascaris-specific antibodies and other factors. Gene expression analysis of serum-activated macrophages revealed that CCL24, a potent eosinophil attractant, was the most upregulated gene following culture with A suum larvae in vitro, and human eosinophils displayed even greater ability to adhere to, and trap, A suum larvae. These data suggest that immune serum-activated macrophages can recruit eosinophils to the site of infection, where they act in concert to immobilize tissue-migrating Ascaris larvae.

Immune Serum From Sabin Inactivated Poliovirus Vaccine Immunization Neutralizes Multiple Individual Wild and Vaccine-Derived Polioviruses.

BACKGROUND: A Sabin strain-based inactivated poliomyelitis vaccine (Sabin-IPV) is the rational option for completely eradicating poliovirus transmission. The neutralizing capacity of Sabin-IPV immune serum to different strains of poliovirus is a key indicator of the clinical protective efficacy of this vaccine. METHODS: Sera collected from 500 infants enrolled in a randomized, blinded, positive control, phase 2 clinical trial were randomly divided into 5 groups: Groups A, B, and C received high, medium, and low doses, respectively, of Sabin-IPV, while groups D and E received trivalent oral polio vaccine and Salk strain-based IPV, respectively, all on the same schedule. Immune sera were collected after the third dose of primary immunization, and tested in cross-neutralization assays against 19 poliovirus strains of all 3 types. RESULTS: All immune sera from all 5 groups interacted with the 19 poliovirus strains with various titers and in a dose-dependent manner. One type 2 immunodeficiency-associated vaccine-derived poliovirus strain was not recognized by these immune sera. CONCLUSIONS: Sabin-IPV vaccine can induce protective antibodies against currently circulating and reference wild poliovirus strains and most vaccine-derived poliovirus strains, with rare exceptions. CLINICAL TRIALS REGISTRATION: NCT01056705.

Common ragweed (Ambrosia artemisiifolia L.): allergenicity and molecular characterization of pollen after plant exposure to elevated NO2.

Ragweed pollen is the main cause of allergenic diseases in Northern America, and the weed has become a spreading neophyte in Europe. Climate change and air pollution are speculated to affect the allergenic potential of pollen. The objective of this study was to investigate the effects of NO2 , a major air pollutant, under controlled conditions, on the allergenicity of ragweed pollen. Ragweed was exposed to different levels of NO2 throughout the entire growing season, and its pollen further analysed. Spectroscopic analysis showed increased outer cell wall polymers and decreased amounts of pectin. Proteome studies using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry indicated increased amounts of several Amb a 1 isoforms and of another allergen with great homology to enolase Hev b 9 from rubber tree. Analysis of protein S-nitrosylation identified nitrosylated proteins in pollen from both conditions, including Amb a 1 isoforms. However, elevated NO2 significantly enhanced the overall nitrosylation. Finally, we demonstrated increased overall pollen allergenicity by immunoblotting using ragweed antisera, showing a significantly higher allergenicity for Amb a 1. The data highlight a direct influence of elevated NO2 on the increased allergenicity of ragweed pollen and a direct correlation with an increased risk for human health.

Structural and functional differences between pheromonotropic and melanotropic PK/PBAN receptors.

BACKGROUND: The pyrokinin/pheromone biosynthesis-activating neuropeptide (PK/PBAN) plays a major role in regulating a wide range of physiological processes in insects. The ubiquitous and multifunctional nature of the PK/PBAN peptide family raises many questions regarding the mechanisms by which these neuropeptides elicit their effects and the nature of the receptors that mediate their functions. METHODS: A sex pheromone gland receptor of the PK/PBAN family from Heliothis peltigera female moth and a Spodoptera littoralis larval receptor were cloned and stably expressed, and their structural models, electrostatic potentials and cellular functional properties were evaluated. RESULTS: Homology modeling indicated highly conserved amino-acid residues in appropriate structural positions as experimentally shown for class A G-protein coupled receptors. Structural differences could be proposed and electrostatic potentials of the two receptor models revealed net charge differences. Calcium mobilization assays demonstrated that both receptors were fully functional and could initiate extracellular calcium influx to start PK/PBAN signal transduction. Evaluation of the signaling response of both receptors to PBAN and diapause hormone (DH) revealed a highly sensitive, though differential response. Both receptors responded to PBAN whereas only Spl-PK/PBAN-R exhibited a high response toward DH. CONCLUSIONS: The structural, electrostatic and cellular functional differences indicate that different PK/PBAN in vivo functions may be mediated by different PK/PBAN receptors and elicited by different peptide(s). GENERAL SIGNIFICANCE: The results advance our understanding of the mode of action of the PK/PBAN family, and might help in exploring novel high-affinity receptor-specific antagonists that can serve as a basis for the development of new families of insect-control agents.

Dynamics of Humoral Immunity to Myxoma and Rabbit Hemorrhagic Disease Viruses in Wild European Rabbits Assessed by Longitudinal Semiquantitative Serology.

Myxoma virus (MYXV) and rabbit hemorrhagic disease virus (RHDV) are important drivers of the population decline of the European rabbit, an endangered keystone species. Both viruses elicit strong immune responses, but the long-term dynamics of humoral immunity are imperfectly known. This study aimed to assess the determinants of the long-term dynamics of antibodies to each virus based on a longitudinal capture-mark-recapture of wild European rabbits and semiquantitative serological data of MYXV and RHDV GI.2-specific IgG. The study included 611 indirect enzyme-linked immunosorbent assay (iELISA) normalized absorbance ratios for each MYXV and RHDV GI.2 from 505 rabbits from 2018 to 2022. Normalized absorbance ratios were analyzed using log-linear mixed models, showing a significant positive relationship with the time since the first capture of individual rabbits, with monthly increases of 4.1% for antibodies against MYXV and 2.0% against RHDV GI.2. Individual serological histories showed fluctuations over time, suggesting that reinfections boosted the immune response and likely resulted in lifelong immunity. Normalized absorbance ratios significantly increased with the seroprevalence in the population, probably because of recent outbreaks, and with body weight, highlighting the role of MYXV and RHDV GI.2 in determining survival to adulthood. Juvenile rabbits seropositive for both viruses were found, and the dynamics of RHDV GI.2 normalized absorbance ratios suggest the presence of maternal immunity up to 2 months of age. Semiquantitative longitudinal serological data provide epidemiological information, otherwise lost when considering only qualitative data, and support a lifelong acquired humoral immunity to RHDV GI.2 and MYXV upon natural infection. IMPORTANCE This study addresses the long-term dynamics of humoral immunity to two major viral pathogens of the European rabbit, an endangered keystone species of major ecological relevance. Such studies are particularly challenging in free-ranging species, and a combination of longitudinal capture-mark-recapture and semiquantitative serology was used to address this question. Over 600 normalized absorbance ratios of iELISA, obtained from 505 individual rabbits in 7 populations over 5 years, were analyzed using linear mixed models. The results support a lifelong acquired humoral immunity to myxoma virus and rabbit hemorrhagic disease virus upon natural infection and suggest the presence of maternal immunity to the latter in wild juvenile rabbits. These results contribute to understanding the epidemiology of two viral diseases threatening this keystone species and assist in developing conservation programs.

Clinical progress of therapeutics and vaccines: Rising hope against COVID-19 treatment.

Cases of deaths due to COVID-19 (COrona VIrus Disease-19) infection are increasing gradually worldwide. Immense research is ongoing to control this pandemic condition. Continual research outcomes are indicating that therapeutic and prophylactic agents are the possible hope to prevent the pandemic from spreading and to combat this increasing death count. Experience gained from previous coronavirus infections (eg., SARS (Severe Acute Respiratory Syndrome), MERS (Middle Ease Respiratory Syndrome), accumulated clinical knowledge during this pandemic, and research helped to identify a few therapeutic agents for emergency treatment of COVID-19. Thereby, monoclonal antibodies, antivirals, broad-spectrum antimicrobials, immunomodulators, and supplements are being suggested for treatment depending on the stage of the disease. These recommended treatments are authorized under medical supervision in emergency conditions only. Urgent need to control the pandemic condition had resulted in various approaches of repurposing the existing drugs, However, poorly designed clinical trials and associated outcomes do not provide enough evidence to fully approve treatments against COVID-19. So far, World Health Organization (WHO) authorized three vaccines as prophylactic against SARS-CoV-2. Here, we discussed about various therapeutic agents, their clinical trials, and limitations of trials for the management of COVID-19. Further, we have also spotlighted different vaccines in research in combating COVID-19.

Cross-neutralization Capacity of Immune Serum from Different Dosage of Sabin Inactivated Poliovirus Vaccine Immunization against Multiple Individual Polioviruses.

Introduction: Sabin strain inactivated poliovirus vaccine (sIPV) developed by Sinovac Biotech Co., Ltd., has shown good safety and immunogenicity against parental strains among infants in several finished pre-licensure clinical trials.Areas covered: To further study the neutralizing capacity of investigational sIPV immune serum against Sabin, Salk and recently circulating poliovirus strains, neutralization assay against ten individual strains was performed on backup serum collected from 250 infant participants of the finished phase II clinical trial.Expert commentary:: The sIPV can generate good immunogenicity against Sabin, Salk and recently circulating poliovirus strains. Taking into account its lower containment requirements and financial costs compared with the conventional Salk strain inactivated poliovirus vaccine, sIPV is an affordable and practical option for polio eradication.

Strategy for the Enrichment of Protein Biomarkers from Diverse Bacterial Select Agents.

BACKGROUND: Some pathogenic bacteria can be potentially used for nefarious applications in the event of bioterrorism or biowarfare. Accurate identification of biological agent from clinical and diverse environmental matrices is of paramount importance for implementation of medical countermeasures and biothreat mitigation. OBJECTIVE: A novel methodology is reported here for the development of a novel enrichment strategy for the generally conserved abundant bacterial proteins for an accurate downstream species identification using tandem MS analysis in biothreat scenario. METHODS: Conserved regions in the common bacterial protein markers were analyzed using bioinformatic tools and stitched for a possible generic immuno-capture for an intended downstream MS/MS analysis. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of 60 kDa chaperonin GroEL. Hyper-immune serum was raised against recombinant synthetic GroEL protein. RESULTS: The conserved regions of common bacterial proteins were stitched for a possible generic immuno-capture and subsequent specific identification by tandem MS using variable regions of the molecule. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of GroEL. In a proof-of-concept study, hyper-immune serum raised against recombinant synthetic GroEL protein exhibited reactivity with ~60 KDa proteins from the cell lysates of three bacterial species tested. CONCLUSION: The envisaged methodology can lead to the development of a novel enrichment strategy for the abundant bacterial proteins from complex environmental matrices for the downstream species identification with increased sensitivity and substantially reduce the time-to-result.

A Brief History of Ebolavirus Disease: Paving the Way Forward by Learning from the Previous Outbreaks.

In this review, Ebolavirus Disease (EVD) outbreaks have been comprehensively reviewed from their beginning until now. It chronologically discusses how each outbreak was tackled, national and international actions taken, diagnostic methods applied, the infection control procedures put in place, and the lessons learnt from each epidemic for the control of subsequent epidemics. Data for this review were obtained from literature published between 1967 and 2016 in key medical databases, the official websites of various governmental organisations, international public health agencies, and regulatory bodies. Despite major developments in the field of EVD, there has been little progress in its specific therapy or prevention. Historically, individuals who recovered from EVD acted as a source of fresh frozen plasma (containing IgG) that has been used to treat other acutely ill patients, however this therapeutic modality has limitations due to the risk of transmission of blood-borne infections. With the use of advanced and efficient purification methods the incidence of unwanted side effects following immune serum therapy has currently been greatly reduced. Creation of a safe plasma pool that covers immunoglobulins against all strains of EVD is now a research priority. Recommendations on how future EVD outbreaks can be better managed have been discussed.

Estimated protective effectiveness of intramuscular immune serum globulin post-exposure prophylaxis during a measles outbreak in British Columbia, Canada, 2014.

INTRODUCTION: Intramuscular Immune Serum Globulin (IM ISG) is recommended as post-measles exposure prophylaxis (PEP) when administered within 6days of initial exposure, with variable effectiveness in preventing measles disease. Effectiveness of IM ISG PEP in preventing clinical measles was assessed during a 2014 measles outbreak among a religious-affiliated community in British Columbia, Canada. MATERIAL AND METHODS: Fifty-five self-reporting measles susceptible contacts were offered exclusively IM ISG PEP within an eligibility period best surmised to be within 6days of initial measles case exposure. Clinical outcome of IM ISG PEP recipients was determined by selective active surveillance and case self-reporting. IM ISG PEP failure was defined as onset of a measles-like rash 8-21days post-IM ISG PEP. Post-IM ISG PEP measles IgG antibody level was tested in 8 recipients. Factors associated with measles disease were analyzed. RESULTS: Seventeen of 55 IM ISG PEP recipients developed clinically consistent measles in the following 8-21days, corresponding to an estimated crude protective effectiveness of 69%. In school aged children 5-18years, among whom potential exposure intensity and immune status confounders were considered less likely, estimated IM ISG PEP protective effectiveness was 50%. Age <25years was significantly associated with breakthrough clinical measles in bivariate analysis (p=0.0217). Among 8 tested contacts of 17 considered IM ISG PEP failures, post-IM ISG PEP measles IgG antibody levels (mean 16.3days (range 16-17days) post-PEP) were all <150mIU/ml. CONCLUSIONS: The estimated crude IM ISG PEP protective effectiveness against measles disease within 8-21days post-ISG administration was 69%. Accuracy of this estimated protective effectiveness is vulnerable to assumptions and uncertainties in ascertaining exposure details and pre-exposure immune status. Increasing the Canadian recommended measles IM ISG PEP dose from 0.25 to 0.5ml/kg (up to 15ml maximum volume) may increase protective effectiveness.

Passive immunotherapy for Middle East Respiratory Syndrome coronavirus infection with equine immunoglobulin or immunoglobulin fragments in a mouse model.

Middle East Respiratory Syndrome (MERS) is a highly lethal pulmonary infection caused by a coronavirus (CoV), MERS-CoV. With the continuing spread of MERS-CoV, prophylactic and therapeutic treatments are urgently needed. In this study, we prepared purified equine F(ab')2 from horses immunized with MERS-CoV virus-like particles (VLPs) expressing MERS-CoV S, M and E proteins. Both IgG and F(ab')2 efficiently neutralized MERS-CoV replication in tissue culture. Passive transfer of equine immune antibodies significantly reduced virus titers and accelerated virus clearance from the lungs of MERS-CoV infected mice. Our data show that horses immunized with MERS-CoV VLPs can serve as a primary source of protective F(ab')2 for potential use in the prophylactic or therapeutic treatment of exposed or infected patients.

The effect of adenovirus-specific antibodies on adenoviral vector-induced, transgene product-specific T cell responses.

In this study, we tested the effect of neutralizing Abs to different serotypes of E1-deleted Ad vectors on the immunogenicity of the homologous Ad vector or a vector derived from a heterologous serotype. Our results showed that, as expected, even low titers of passively transferred neutralizing Abs significantly reduced the homologous vectors' ability to elicit transgene-specific CD8(+) T cell responses. In addition, Abs changed the fate of transgene product-specific CD8(+) T cells by promoting their transition into the central memory cell pool, which resulted in markedly enhanced expansion of transgene product-specific CD8(+) T cells after a boost with a heterologous Ad vector. Non-neutralizing Abs specific to a distinct Ad serotype had no effect on the magnitude of transgene product-specific CD8(+) T cells induced by a heterologous Ad vector, nor did such Abs promote induction of more resting memory CD8(+) T cells. These results show that Abs to an Ad vaccine carrier affect not only the magnitude but also the profile of a vector-induced CD8(+) T cell response.

Prevention of cholestasis in the murine rotavirus-induced biliary atresia model using passive immunization and nonreplicating virus-like particles.

Biliary atresia (BA) is a neonatal cholangiopathy of unknown etiology that results in obliteration of bile ducts and is the primary indication for liver transplant in children. A murine model of BA, which involves infecting newborn mice with rhesus rotavirus (RRV) and leads to development of an obstructive cholangiopathy, has provided a model to assess measures to prevent and treat BA. We used this mouse model of RRV-induced BA to determine if passive immunization of pups using maternal immunization [injection of dams with non-replicating rotavirus (RV) virus-like particles (VLPs) or live RRV] or injection of pups with RV immune serum would protect these RRV-infected neonates from developing BA (measured using cholestasis). Parenteral immunization of mouse dams with two formulations of VLPs (containing viral proteins 2/6 or 2/6/7) resulted in a significant increase in serum RV antibody, and pups born to these immunized dams were protected from developing cholestasis following neonatal infection with RRV. Serum RV-specific antibody with titers of ≥400-800 in dams significantly protected pups from developing cholestasis, and a significant trend of increasing protection with high titers was observed (p<0.0001). Cholestatic pups had lower levels of RV serum antibody and higher serum (p<0.01) and liver (p<0.05) RV antigen compared to healthy pups. Passive transfer of low-titer (1600; p<0.05) or high-titer (25,600; p<0.01) RV immune serum to neonatal pups prior to RRV infection also protected them from developing cholestasis. Together, these findings indicate that passively acquired, neutralizing or non-neutralizing RV serum antibody attenuates viral replication and protects pups against disease in the RRV BA model. Early reduction of viral load by clearance with RV-specific antibody is likely a critical determinant of disease in this model.