Purification and biochemical characterization of four iron superoxide dismutases in Trypanosoma cruzi

Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi . The total extract was subjected to two successive ammonium sulphate additions between 35 and 85%, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low- mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.Financial support: CGL2006-27889-E/BOS, Ministerio de Ciencia y Tecnología

Stalking influenza by vaccination with pre-fusion headless HA mini-stem.

Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies indudced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity

Emerging Technologies for the Detection of Rabies Virus: Challenges and Hopes in the 21st Century

The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human rabies is now possible using molecular techniques. These barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis

Llama-Derived Single Domain Antibodies to Build Multivalent, Superpotent and Broadened Neutralizing Anti-Viral Molecules

For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC50 of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5. The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve "best-in-class" and broader neutralization capacity

The Trypanosoma cruzi vitamin C dependent peroxidase confers protection against oxidative stress but is not a determinant of virulence.

BACKGROUND: The neglected parasitic infection Chagas disease is rapidly becoming a globalised public health issue due to migration. There are only two anti-parasitic drugs available to treat this disease, benznidazole and nifurtimox. Thus it is important to identify and validate new drug targets in Trypanosoma cruzi, the causative agent. T. cruzi expresses an ER-localised ascorbate-dependent peroxidase (TcAPx). This parasite-specific enzyme has attracted interest from the perspective of targeted chemotherapy. METHODOLOGY/PRINCIPAL FINDINGS: To assess the importance of TcAPx in protecting T. cruzi from oxidative stress and to determine if it is essential for virulence, we generated null mutants by targeted gene disruption. Loss of activity was associated with increased sensitivity to exogenous hydrogen peroxide, but had no effect on susceptibility to the front-line Chagas disease drug benznidazole. This suggests that increased oxidative stress in the ER does not play a significant role in its mechanism of action. Homozygous knockouts could proceed through the entire life-cycle in vitro, although they exhibited a significant decrease in their ability to infect mammalian cells. To investigate virulence, we exploited a highly sensitive bioluminescence imaging system which allows parasites to be monitored in real-time in the chronic stage of murine infections. This showed that depletion of enzyme activity had no effect on T. cruzi replication, dissemination or tissue tropism in vivo. CONCLUSIONS/SIGNIFICANCE: TcAPx is not essential for parasite viability within the mammalian host, does not have a significant role in establishment or maintenance of chronic infections, and should therefore not be considered a priority for drug design

Cross-Neutralizing Antibodies to Pandemic 2009 H1N1 and Recent Seasonal H1N1 Influenza A Strains Influenced by a Mutation in Hemagglutinin Subunit 2

Pandemic 2009 H1N1 influenza A virus (2009 H1N1) differs from H1N1 strains that circulated in the past 50 years, but resembles the A/New Jersey/1976 H1N1 strain used in the 1976 swine influenza vaccine. We investigated whether sera from persons immunized with the 1976 swine influenza or recent seasonal influenza vaccines, or both, neutralize 2009 H1N1. Using retroviral pseudovirions bearing hemagglutinins on their surface (HA-pseudotypes), we found that 77% of the sera collected in 1976 after immunization with the A/New Jersey/1976 H1N1 swine influenza vaccine neutralized 2009 H1N1. Forty five percent also neutralized A/New Caledonia/20/1999 H1N1, a strain used in seasonal influenza vaccines during the 2000/01–2006/07 seasons. Among adults aged 48–64 who received the swine influenza vaccine in 1976 and recent seasonal influenza vaccines during the 2004/05–2008/09 seasons, 83% had sera that neutralized 2009 H1N1. However, 68% of age-matched subjects who received the same seasonal influenza vaccines, but did not receive the 1976 swine influenza vaccine, also had sera that neutralized 2009 H1N1. Sera from both 1976 and contemporary cohorts frequently had cross-neutralizing antibodies to 2009 H1N1 and A/New Caledonia/20/1999 that mapped to hemagglutinin subunit 2 (HA2). A conservative mutation in HA2 corresponding to a residue in the A/Solomon Islands/3/2006 and A/Brisbane/59/2007 H1N1 strains that circulated in the 2006/07 and 2007/08 influenza seasons, respectively, abrogated this neutralization. These findings highlight a cross-neutralization determinant influenced by a point mutation in HA2 and suggest that HA2 may be evolving under direct or indirect immune pressure

A naturally protective epitope of limited variability as an influenza vaccine target

Current antigenic targets for influenza vaccine development are either highly immunogenic epitopes of high variability or conserved epitopes of low immunogenicity. This requires continuous update of the variable epitopes in the vaccine formulation or boosting of immunity to invariant epitopes of low natural efficacy. Here we identify a highly immunogenic epitope of limited variability in the head domain of the H1 haemagglutinin protein. We show that a cohort of young children exhibit natural immunity to a set of historical influenza strains which they could not have previously encountered and that this is partially mediated through the epitope. Furthermore, vaccinating mice with these epitope conformations can induce immunity to human H1N1 influenza strains that have circulated since 1918. The identification of epitopes of limited variability offers a mechanism by which a universal influenza vaccine can be created; these vaccines would also have the potential to protect against newly emerging influenza strains

The impact of viral mutations on recognition by SARS-CoV-2 specific T cells.

We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.This work is supported by the UK Medical Research Council (MRC); Chinese Academy of Medical Sciences(CAMS) Innovation Fund for Medical Sciences (CIFMS), China; National Institute for Health Research (NIHR)Oxford Biomedical Research Centre, and UK Researchand Innovation (UKRI)/NIHR through the UK Coro-navirus Immunology Consortium (UK-CIC). Sequencing of SARS-CoV-2 samples and collation of data wasundertaken by the COG-UK CONSORTIUM. COG-UK is supported by funding from the Medical ResearchCouncil (MRC) part of UK Research & Innovation (UKRI),the National Institute of Health Research (NIHR),and Genome Research Limited, operating as the Wellcome Sanger Institute. T.I.d.S. is supported by a Well-come Trust Intermediate Clinical Fellowship (110058/Z/15/Z). L.T. is supported by the Wellcome Trust(grant number 205228/Z/16/Z) and by theUniversity of Liverpool Centre for Excellence in Infectious DiseaseResearch (CEIDR). S.D. is funded by an NIHR GlobalResearch Professorship (NIHR300791). L.T. and S.C.M.are also supported by the U.S. Food and Drug Administration Medical Countermeasures Initiative contract75F40120C00085 and the National Institute for Health Research Health Protection Research Unit (HPRU) inEmerging and Zoonotic Infections (NIHR200907) at University of Liverpool inpartnership with Public HealthEngland (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.L.T. is based at the University of Liverpool. M.D.P. is funded by the NIHR Sheffield Biomedical ResearchCentre (BRC – IS-BRC-1215-20017). ISARIC4C is supported by the MRC (grant no MC_PC_19059). J.C.K.is a Wellcome Investigator (WT204969/Z/16/Z) and supported by NIHR Oxford Biomedical Research Centreand CIFMS. The views expressed are those of the authors and not necessarily those of the NIHR or MRC