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Background: Administration of potent anti-receptor-binding domain (RBD) monoclonal antibodies has been shown to curtail viral shedding and reduce hospitalization in patients with SARS-CoV-2 infection. However, the structure-function analysis of potent human anti-RBD monoclonal antibodies and its links to the formulation of antibody cocktails remains largely elusive. Methods: Previously, we isolated a panel of neutralizing anti-RBD monoclonal antibodies from convalescent patients and showed their neutralization efficacy in vitro. Here, we elucidate the mechanism of action of antibodies and dissect antibodies at the epitope level, which leads to a formation of a potent antibody cocktail. Results: We found that representative antibodies which target non-overlapping epitopes are effective against wild type virus and recently emerging variants of concern, whilst being encoded by antibody genes with few somatic mutations. Neutralization is associated with the inhibition of binding of viral RBD to ACE2 and possibly of the subsequent fusion process. Structural analysis of representative antibodies, by cryo-electron microscopy and crystallography, reveals that they have some unique aspects that are of potential value while sharing some features in common with previously reported neutralizing monoclonal antibodies. For instance, one has a common VH 3-53 public variable region yet is unusually resilient to mutation at residue 501 of the RBD. We evaluate the in vivo efficacy of an antibody cocktail consisting of two potent non-competing anti-RBD antibodies in a Syrian hamster model. We demonstrate that the cocktail prevents weight loss, reduces lung viral load and attenuates pulmonary inflammation in hamsters in both prophylactic and therapeutic settings. Although neutralization of one of these antibodies is abrogated by the mutations of variant B.1.351, it is also possible to produce a bi-valent cocktail of antibodies both of which are resilient to variants B.1.1.7, B.1.351 and B.1.617.2. Conclusions: These findings support the up-to-date and rational design of an anti-RBD antibody cocktail as a therapeutic candidate against COVID-19.

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection., Neutralizing nanobodies (Nb) are of considerable interest as therapeutic agents for COVID-19 treatment. Here, the authors functionally and structurally characterize Nbs that bind with high affinity to the receptor binding domain of the SARS-CoV-2 spike protein and show that an engineered homotrimeric Nb prevents disease progression in a Syrian hamster model of COVID-19 when administered intranasally.

Phosphodiesterase 12 (PDE12) is a negative regulator of the type 1 interferon (IFN) response and here we show that PDE12 inhibitors (lead compounds 63 and 17) are associated with increased RNAseL activity, are well tolerated at the therapeutic range and inhibit, both in vitro and in vivo, the replication of several RNA viruses including hepatitis C virus (HCV), dengue virus (DENV), West Nile Virus (WNV) and SARS-CoV-2.

Antibodies against SARS-CoV-2 are important to generate protective immunity, with convalescent plasma one of the first therapies approved. An alternative source of polyclonal antibodies suitable for upscaling would be more amendable to regulatory approval and widespread use. In this study, sheep were immunised with SARS-CoV-2 whole spike protein or one of the subunit proteins: S1 and S2. Once substantial antibody titres were generated, plasma was collected and samples pooled for each antigen. Non-specific antibodies were removed via affinity-purification to yield candidate products for testing in a hamster model of SARS-CoV-2 infection. Affinity-purified polyclonal antibodies to whole spike, S1 and S2 proteins were evaluated for in vitro for neutralising activity against SARS-CoV-2 Wuhan-like virus (Australia/VIC01/2020) and a recent variant of concern, B.1.1.529 BA.1 (Omicron), antibody-binding, complement fixation and phagocytosis assays were also performed. All antibody preparations demonstrated an effect against SARS-CoV-2 disease in the hamster model of challenge, with those raised against the S2 subunit providing the most promise. A rapid, cost-effective therapy for COVID-19 was developed which provides a source of highly active immunoglobulin specific to SARS-CoV-2 with multi-functional activity.

A novel coronavirus, SARS-CoV-2, has been identified as the causative agent of the current COVID-19 pandemic. Animal models, and in particular non-human primates, are essential to understand the pathogenesis of emerging diseases and to assess the safety and efficacy of novel vaccines and therapeutics. Here, we show that SARS-CoV-2 replicates in the upper and lower respiratory tract and causes pulmonary lesions in both rhesus and cynomolgus macaques. Immune responses against SARS-CoV-2 are also similar in both species and equivalent to those reported in milder infections and convalescent human patients. This finding is reiterated by our transcriptional analysis of respiratory samples revealing the global response to infection. We describe a new method for lung histopathology scoring that will provide a metric to enable clearer decision making for this key endpoint. In contrast to prior publications, in which rhesus are accepted to be the preferred study species, we provide convincing evidence that both macaque species authentically represent mild to moderate forms of COVID-19 observed in the majority of the human population and both species should be used to evaluate the safety and efficacy of interventions against SARS-CoV-2. Importantly, accessing cynomolgus macaques will greatly alleviate the pressures on current rhesus stocks., Non-human primates are important animal models for studying SARS-CoV-2 infection. Here, Salguero et al. directly compare rhesus and cynomolgus macaques and show that both species represent COVID-19 disease of mild clinical cases, and provide a lung histopathology scoring system.

There is need for effective and affordable vaccines against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a protein nanoparticle vaccine against SARS-CoV-2. The vaccine is based on the display of coronavirus spike glycoprotein receptor-binding domain (RBD) on a synthetic virus-like particle (VLP) platform, SpyCatcher003-mi3, using SpyTag/SpyCatcher technology. Low doses of RBD-SpyVLP in a prime-boost regimen induce a strong neutralising antibody response in mice and pigs that is superior to convalescent human sera. We evaluate antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we show that RBD-SpyVLP induces a polyclonal antibody response that recognises key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. Moreover, RBD-SpyVLP is thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence. The data suggests that RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic., Vaccines for SARS-COV-2 are needed in the ongoing pandemic. Here the authors characterize a vaccine candidate that presents the receptor-binding domain (RBD) of SARS-CoV-2 spike protein on a synthetic VLP platform using SpyTag/SpyCatcher technology and show immunogenicity of a prime-boost regimen in mice and pigs.

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity, or as a therapeutic, has yet been developed to SARS-CoV-2. In this study, we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks ACE2 receptor binding, by overlapping the ACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in 293 cells expressing ACE2. When converted to secretory IgA, MAb326 also neutralizes authentic SARS-CoV-2 virus while the IgG isotype shows no neutralization. Our results suggest that SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

Filoviruses cause high-consequence infections with limited approved medical countermeasures (MCMs). MCM development is dependent upon well-characterized animal models for the assessment of antiviral agents and vaccines. Following large-scale Ebola virus (EBOV) disease outbreaks in Africa, some survivors are left with long-term sequelae and persistent virus in immune-privileged sites for many years. We report the characterization of the ferret as a model for Ebola virus infection, reproducing disease and lethality observed in humans. The onset of clinical signs is rapid, and EBOV is detected in the blood, oral, and rectal swabs and all tissues studied. We identify viral RNA in the eye (a site of immune privilege) and report on specific genomic changes in EBOV present in this structure. Thus, the ferret model has utility in testing MCMs that prevent or treat long-term EBOV persistence in immune-privileged sites. IMPORTANCE Recent reemergence of Ebola in Guinea that caused over 28,000 cases between 2013 and 2016 has been linked to the original virus from that region. It appears the virus has remained in the region for at least 5 years and is likely to have been maintained in humans. Persistence of Ebola in areas of the body for extended periods of time has been observed, such as in the eye and semen. Despite the importance of reintroduction of Ebola from this route, such events are rare in the population, which makes studying medical interventions to clear persistent virus difficult. We studied various doses of Ebola in ferrets and detected virus in the eyes of most ferrets. We believe this model will enable the study of medical interventions that promote clearance of Ebola virus from sites that promote persistence.

Aims The goal of this study was to examine, for the first time, the virulence and pathogenicity of aerosolized Burkholderia pseudomallei, strain NCTC 13392, in BALB/c mice in order to develop an animal model for testing novel medical countermeasures (MCMs) for the treatment of human acute and subacute (a disease state between acute and chronic) melioidosis. Methods and Results BALB/c mice were exposed to varying doses of aerosolized bacteria. Acute disease was seen in animals exposed to a very‐high dose (≥103 CFU per animal) and death occurred 3–4 days postchallenge (pc). Bacteria were detected in the lungs, liver, kidney and spleen. In contrast, animals exposed to a low dose (

IntroductionSARS-CoV-2 has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgRNAs has a unique 5’ sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS-junction), that can be identified using sequencing.ResultsHigh resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS-junctions and be used as a proxy to quantify sgmRNAs for understanding virus biology. This was tested on published datasets and clinical samples from patients and longitudinal samples from animal models with COVID-19.DiscussionLeTRS identified known leader-TRS-junctions and identified novel species that were common across different species. The data indicated multi-phasic abundance of sgmRNAs in two different animal models, with spikes in sgmRNA abundance reflected in human samples, and therefore has implications for transmission models and nucleic acid-based diagnostics.

Background and Purpose: Currently, there are no licensed vaccines and limited antivirals for the treatment of COVID-19. Heparin (delivered systemically) is currently used to treat anticoagulant anomalies in COVID-19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is being trialled in COVID-19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild type SARS-CoV-2, in vitro, is needed. Experimental Approach: Seven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS-CoV-2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined. Key Results: All the UFH preparations had potent antiviral effects, with IC50 values ranging between 25 and 41 μg·ml−1, whereas LMWHs were less inhibitory by ~150-fold (IC50 range 3.4–7.8 mg·ml−1). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor. Conclusion and Implications: This comparison of clinically relevant heparins shows that UFH has significantly stronger SARS-CoV-2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID-19.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5–15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease., SARS-CoV-2 induces mild infection in ferret model. Here, Ryan et al. characterise optimal infection dosage inducing upper respiratory tract (UTR) viral shedding, progression time of viral shedding, and pathology in ferrets and finally provide evidence for protection after re-challenge.

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

Background: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. Methods: We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18–55 years with no history of laboratory confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 × 10 10 viral particles or MenACWY as a single intramuscular injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque reduction neutralisation assay [PRNT 50]; a microneutralisation assay [MNA 50, MNA 80, and MNA 90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. Findings: Between April 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p80 and in 35 (100%) participants when measured in PRNT 50. After a booster dose, all participants had neutralising activity (nine of nine in MNA 80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R 2=0·67 by Marburg VN; p

The COVID-19 pandemic has had unprecedented health and economic impact, but currently there are no approved therapies. We have isolated an antibody, EY6A, from a late-stage COVID-19 patient and show it neutralises SARS-CoV-2 and cross-reacts with SARS-CoV-1. EY6A Fab binds tightly (KD of 2 nM) the receptor binding domain (RBD) of the viral Spike glycoprotein and a 2.6Å crystal structure of an RBD/EY6A Fab complex identifies the highly conserved epitope, away from the ACE2 receptor binding site. Residues of this epitope are key to stabilising the pre-fusion Spike. Cryo-EM analyses of the pre-fusion Spike incubated with EY6A Fab reveal a complex of the intact trimer with three Fabs bound and two further multimeric forms comprising destabilized Spike attached to Fab. EY6A binds what is probably a major neutralising epitope, making it a candidate therapeutic for COVID-19.

Nebulised unfractionated heparin (UFH) has a strong scientific and biological rationale and warrants urgent investigation of its therapeutic potential, for COVID-19-induced acute respiratory distress syndrome (ARDS). COVID-19 ARDS displays the typical features of diffuse alveolar damage with extensive pulmonary coagulation activation resulting in fibrin deposition in the microvasculature and formation of hyaline membranes in the air sacs. Patients infected with SARS-CoV-2 who manifest severe disease have high levels of inflammatory cytokines in plasma and bronchoalveolar lavage fluid and significant coagulopathy. There is a strong association between the extent of the coagulopathy and poor clinical outcomes.The anti-coagulant actions of nebulised UFH limit fibrin deposition and microvascular thrombosis. Trials in patients with acute lung injury and related conditions found inhaled UFH reduced pulmonary dead space, coagulation activation, microvascular thrombosis and clinical deterioration, resulting in increased time free of ventilatory support. In addition, UFH has anti-inflammatory, mucolytic and anti-viral properties and, specifically, has been shown to inactivate the SARS-CoV-2 virus and prevent its entry into mammalian cells, thereby inhibiting pulmonary infection by SARS-CoV-2. Furthermore, clinical studies have shown that inhaled UFH safely improves outcomes in other inflammatory respiratory diseases and also acts as an effective mucolytic in sputum-producing respiratory patients. UFH is widely available and inexpensive, which may make this treatment also accessible for low- and middle-income countries.These potentially important therapeutic properties of nebulised UFH underline the need for expedited large-scale clinical trials to test its potential to reduce mortality in COVID-19 patients.

Background and Purpose: Currently there are no licensed vaccines and limited antivirals for the treatment of COVID-19. Heparin (delivered systemically) is currently being used to treat anticoagulant anomalies in COVID-19 patients. In addition, in the UK, nebulised unfractionated heparin (UFH) is currently being trialled in COVID-19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild-type SARS-CoV-2, in vitro, is thus urgently needed. Experimental Approach: A range of heparin preparations both UFH (n=4) and low molecular weight heparins (LMWH) (n=3) of porcine or bovine origin were screened for antiviral activity against live SARS-CoV-2 (Victoria/01/2020) using a plaque reduction neutralisation assay and Vero E6 cells. ND50 values for each heparin were calculated using a mid-point probit analysis. Key Results: UFH had potent antiviral effects, with ND50 values of 12.5 and 23 μg/ml for two porcine mucosal UFH tested. Bovine mucosal UFH had similar antiviral effects although it was ~50% less active (ND50, 50-75 μg/ml). In contrast, LMWHs such as Clexane and Fragmin were markedly less active by ~100-fold (ND50 values of 2.6-6.8 mg/ml). Conclusions and Implications: This comparison of a panel of clinically relevant heparins, including the UFH preparation under trial in the UK, demonstrated that distinct products exhibit different degrees of antiviral activity against live SARS-CoV-2. Porcine mucosal UFH has the strongest antiviral activity followed by bovine mucosal UFH, whereas LMWHs had the lowest amount of antiviral activity (by 100-fold). Overall the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID-19.

SummaryHeparan sulfate (HS) is a cell surface polysaccharide recently identified as a co-receptor with the ACE2 protein for recognition of the S1 spike protein on SARS-CoV-2 virus, providing a tractable new target for therapeutic intervention. Clinically-used heparins demonstrate inhibitory activity, but world supplies are limited, necessitating alternative solutions. Synthetic HS mimetic pixatimod is a drug candidate for cancer with immunomodulatory and heparanase-inhibiting properties. Here we show that pixatimod binds to and destabilizes the SARS-CoV-2 spike protein receptor binding domain (S1-RBD), and directly inhibits its binding to human ACE2, consistent with molecular modelling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of live SARS-CoV-2 virus show that pixatimod potently inhibits infection of monkey Vero E6 and human bronchial epithelial cells at concentrations within its safe therapeutic dose range. Furthermore, in a K18-hACE2 mouse model pixatimod demonstrates that pixatimod markedly attenuates SARS-CoV-2 viral titer and COVID-19-like symptoms. This demonstration of potent anti-SARS-CoV-2 activity establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics. Together with other known activities of pixatimod our data provides a strong rationale for its clinical investigation as a potential multimodal therapeutic to address the COVID-19 pandemic.

In December 2019 an outbreak of coronavirus disease (COVID-19) emerged in Wuhan, China. The causative agent was subsequently identified and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread worldwide causing a pandemic. Currently there are no licensed vaccines or therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in development and repurposed drugs are being tested in the clinic. There is a vital need for authentic COVID-19 animal models to further our understanding of pathogenesis and viral spread in addition to pre-clinical evaluation of candidate interventions.Here we report a dose titration study of SARS-CoV-2 to determine the most suitable infectious dose to use in the ferret model. We show that a high (5×106 pfu) and medium (5×104 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral RNA shedding in both groups of six challenged animals, whilst a low dose (5×102 pfu) resulted in only one of six displaying signs of URT viral RNA replication. The URT shedding lasted up to 21 days in the high dose animals with intermittent positive signal from day 14. Sequential culls revealed distinct pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were observed although animals did present with a consistent post-viral fatigue lasting from day 9-14 in the medium and high dose groups. After virus shedding ceased, re-challenged ferrets were shown to be fully protected from acute lung pathology. The endpoints of URT viral RNA replication in addition to distinct lung pathology and post viral fatigue were observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 14-21 parallel observations reported in a minority of clinical cases.

The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than influenza. The SARS-CoV-2 receptor binding domain (RBD) of the spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naive llama single-domain antibody library and PCR-based maturation, we have produced two closely related nanobodies, H11-D4 and H11-H4, that bind RBD (KD of 39 and 12 nM, respectively) and block its interaction with ACE2. Single-particle cryo-EM revealed that both nanobodies bind to all three RBDs in the spike trimer. Crystal structures of each nanobody-RBD complex revealed how both nanobodies recognize the same epitope, which partly overlaps with the ACE2 binding surface, explaining the blocking of the RBD-ACE2 interaction. Nanobody-Fc fusions showed neutralizing activity against SARS-CoV-2 (4-6 nM for H11-H4, 18 nM for H11-D4) and additive neutralization with the SARS-CoV-1/2 antibody CR3022.

Summary There are as yet no licensed therapeutics for the COVID-19 pandemic. The causal coronavirus (SARS-CoV-2) binds host cells via a trimeric spike whose receptor binding domain (RBD) recognizes angiotensin-converting enzyme 2, initiating conformational changes that drive membrane fusion. We find that the monoclonal antibody CR3022 binds the RBD tightly, neutralizing SARS-CoV-2, and report the crystal structure at 2.4 Å of the Fab/RBD complex. Some crystals are suitable for screening for entry-blocking inhibitors. The highly conserved, structure-stabilizing CR3022 epitope is inaccessible in the prefusion spike, suggesting that CR3022 binding facilitates conversion to the fusion-incompetent post-fusion state. Cryogenic electron microscopy (cryo-EM) analysis confirms that incubation of spike with CR3022 Fab leads to destruction of the prefusion trimer. Presentation of this cryptic epitope in an RBD-based vaccine might advantageously focus immune responses. Binders at this epitope could be useful therapeutically, possibly in synergy with an antibody that blocks receptor attachment., Graphical Abstract, Highlights • CR3022 binds the RBD of SARS-CoV-2 and shows strong neutralization • Neutralization is by destroying the prefusion spike conformation • CR3022 binds a highly conserved epitope that is inaccessible in prefusion spike protein • CR3022 could have therapeutic potential alone or in synergy with a receptor blocker, Huo et al. find that the antibody CR3022 binds tightly to the receptor binding domain of the SARS-CoV-2 spike at a site different to that used by the receptor. CR3022 effectively neutralizes the virus, and cryo-EM reveals that it disrupts the spike. Such antibodies could have potential as COVID-19 therapeutics.

Understanding the interactions between host and pathogen is important for the development and assessment of medical countermeasures to infectious agents, including potential biodefence pathogens such asBacillus anthracis,Ebola virus, andFrancisella tularensis. This review focuses on technological advances which allow this interaction to be studied in much greater detail. Namely, the use of “omic” technologies (next generation sequencing, DNA, and protein microarrays) for dissecting the underlying host response to infection at the molecular level; optical imaging techniques (flow cytometry and fluorescence microscopy) for assessing cellular responses to infection; and biophotonic imaging for visualising the infectious disease process. All of these technologies hold great promise for important breakthroughs in the rational development of vaccines and therapeutics for biodefence agents.

Aims: We sought to develop a new method that enables the assessment of the immune response of guinea pigs during TB vaccine evaluation studies, without the need to cull or anaesthetize animals. Method and Results: Guinea pigs were vaccinated with five different formulations of oral BCG. One week prior to challenge with Mycobacterium bovis, blood (50–200 μl) was taken from the ears of vaccinated subjects. Host RNA was isolated and amplified following antigenic restimulation of PBMCs for 24 h with 30 μg of bovine PPD. The up- or down-regulation of γ-interferon (IFN-γ), a key cytokine involved in protection against tuberculosis, was assessed using real-time PCR. The relative expression of prechallenge IFN-γ mRNA in the vaccinated groups (n = 5) correlated (P

Iron is an essential cofactor for both mycobacterial growth during infection and for a successful protective immune response by the host. The immune response partly depends on the regulation of iron by the host, including the tight control of expression of the iron-storage protein, ferritin. BCG vaccination can protect against disease following Mycobacterium tuberculosis infection, but the mechanisms of protection remain unclear. To further explore these mechanisms, splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR ( P ≤ 0.05 and ≤0.01, respectively). The mechanisms of this down-regulation were shown to involve TNFα and nitric oxide. A more in depth analysis of the mRNA expression profiles, including genes involved in iron metabolism, was performed using a guinea pig specific immunological microarray following ex vivo infection with M. tuberculosis of splenocytes from BCG-vaccinated and naive guinea pigs. M. tuberculosis infection induced a pro-inflammatory response in splenocytes from both groups, resulting in down-regulation of ferritin ( P ≤ 0.05). In addition, lactoferrin ( P ≤ 0.002), transferrin receptor ( P ≤ 0.05) and solute carrier family 11A1 ( P ≤ 0.05), were only significantly down-regulated after infection of the splenocytes from BCG-vaccinated animals. The results show that expression of iron-metabolism genes is tightly regulated as part of the host response to M. tuberculosis infection and that BCG-vaccination enhances the ability of the host to mount an iron-restriction response which may in turn help to combat invasion by mycobacteria.

Mycobacterium bovis BCG-vaccination in the guinea pig model of tuberculosis (TB) is sufficiently protec-tive that candidate TB vaccines are judged against this. Little is understood about how the BCG vaccineworksand,intheabsenceofadefinitivecorrelateofprotection,itisdifficulttointerpretthesignificanceofnovel vaccine induced host responses. Here an extended custom-made microarray (86 guinea pig genes)wasusedtodissecttemporalchangesinBCG-vaccineinducedgenesignaturestodifferentmycobacterialantigens. Initially at 4h, pro-inflammatory genes such as IL-1 , IL-1 , IL-8 and GRO were up-regulated(P

To evaluate new vaccines when human efficacy studies are not possible, the FDA's “Animal Rule” requires well-characterized models of infection. Thus, in the present study, the early pathogenic events of monkeypox infection in nonhuman primates, a surrogate for variola virus infection, were characterized. Cynomolgus macaques were exposed to aerosolized monkeypox virus (10 5 PFU). Clinical observations, viral loads, immune responses, and pathological changes were examined on days 2, 4, 6, 8, 10, and 12 postchallenge. Viral DNA (vDNA) was detected in the lungs on day 2 postchallenge, and viral antigen was detected, by immunostaining, in the epithelium of bronchi, bronchioles, and alveolar walls. Lesions comprised rare foci of dysplastic and sloughed cells in respiratory bronchioles. By day 4, vDNA was detected in the throat, tonsil, and spleen, and monkeypox antigen was detected in the lung, hilar and submandibular lymph nodes, spleen, and colon. Lung lesions comprised focal epithelial necrosis and inflammation. Body temperature peaked on day 6, pox lesions appeared on the skin, and lesions, with positive immunostaining, were present in the lung, tonsil, spleen, lymph nodes, and colon. By day 8, vDNA was present in 9/13 tissues. Blood concentrations of interleukin 1ra (IL-1ra), IL-6, and gamma interferon (IFN-γ) increased markedly. By day 10, circulating IgG antibody concentrations increased, and on day 12, animals showed early signs of recovery. These results define early events occurring in an inhalational macaque monkeypox infection model, supporting its use as a surrogate model for human smallpox. IMPORTANCE Bioterrorism poses a major threat to public health, as the deliberate release of infectious agents, such smallpox or a related virus, monkeypox, would have catastrophic consequences. The development and testing of new medical countermeasures, e.g., vaccines, are thus priorities; however, tests for efficacy in humans cannot be performed because it would be unethical and field trials are not feasible. To overcome this, the FDA may grant marketing approval of a new product based upon the “Animal Rule,” in which interventions are tested for efficacy in well-characterized animal models. Monkeypox virus infection of nonhuman primates (NHPs) presents a potential surrogate disease model for smallpox. Previously, the later stages of monkeypox infection were defined, but the early course of infection remains unstudied. Here, the early pathogenic events of inhalational monkeypox infection in NHPs were characterized, and the results support the use of this surrogate model for testing human smallpox interventions.

This special issue, dealing with biodefence vaccines and therapies, incorporates two review papers and six original research papers. The review papers introduce concepts and themes underpinning the research papers, including the use of animal models to predict human responses in the transition of candidate vaccines and therapies from research to development and the impact of advances in genomic and imaging technologies on assessing the host immune response to biodefence agents. Biothreat agents usually cause severe disease so clinical efficacy studies of candidate vaccines or therapies will generally not be ethical or feasible, unless in a field trial situation in a region of endemic disease. Thus there is a need to develop authentic models of the human disease in relevant in vitro and ex vivo cell culture and in vivo animal models and to apply these models to evaluate candidates. This process has been facilitated by the publication of the Animal Rule by the US Food and Drug Agency in 2002, through which a number of antibiotics have recently been approved for inhalational anthrax or plague indications, as have therapies for botulism and anthrax and a next generation vaccine for smallpox. More details about this can be found in the review by E. D. Williamson. Biodefense agents are dangerous pathogens that pose unique challenges for researchers who often have to work at high biocontainment (levels 3 and 4) making routine experiments difficult to perform. By using various “state of the art” techniques such as optical imaging, biophotonic imaging, next generation sequencing, and microarrays, data derived from each experiment are greatly enhanced and the study of the host response to infection significantly improved. Recent progress using these “omic” and imaging technologies in biodefense research is reviewed by J. A. Tree et al. Biodefense research benefits from improving and enhancing the utility of specific animal models. In this issue, M. Nelson and M. Loveday describe how they explored the innate immunological response of the common marmoset (Callithrix jacchus) to bacterial challenge. The immune response of this nonhuman primate is often understudied because of a lack of reagents. However, M. Nelson and M. Loveday examined the cross-reactivity of commercially available human reagents with marmoset markers and showed that it was possible to characterise the marmoset immune response to infectious disease in terms of phenotype, cell activation status, and key cytokine and chemokine expression. This is particularly useful because there is an increasing body of evidence that suggests that the physiological and immunological responses to biological agents are similar between marmosets and humans. Progress in the approval of candidate vaccines and therapies is supported by ongoing research which improves the collective understanding of the pathogenesis of disease; two papers in this issue support this area. J. L. Dankmeyer et al. present results about the innate host response to Yersinia pestis. They suggest that Toll-like receptor 4 and the adaptor protein Myd88 are important for an optimal antibody response to the subunit F1-V plague vaccine and that Myd88 also appears to be required for protection against a lethal challenge in vaccinated mice. S. L. Newstead et al. describe the role of nitric oxide in the control of intracellular infection with F. tularensis. They infected two murine macrophage cell lines and demonstrated a differential ability to control the intracellular replication of the attenuated live vaccine strain of F. tularensis compared with the fully virulent SCHU S4 strain. Some control of SCHU S4 replication was achieved by preactivation of macrophages, indicating the involvement of factors additional to NO production in this effect. In order for medical countermeasures to be licensed using the Animal Rule there is a need to bridge human immune responses to the animal models to identify surrogate markers of efficacy. This process is highlighted in the research paper modelling pneumonic plague, by V. A. Graham et al., in which an optimised murine model is described. Serum taken from human volunteers vaccinated with a new acellular plague vaccine (containing recombinant proteins rF1 and rV) was assessed for protective efficacy by passive transfer into mice. Mice were subsequently exposed to a lethal aerosol of plague and the delay in mean time-to-death measured. This process was shown to be reproducible and these data indicated that Hsd:NIHS mice may be a better model than BALB/c mice for passive transfer studies with human serum. The last two papers in this special issue describe research on a generic DNA vaccination approach and a potential immunotherapy treatment for Ebola. S. Qiu and colleagues constructed DNA and recombinant Tiantan vaccinia (rTTV) vaccines encoding OVA-CTB fusion protein. They concluded that, following studies in mice, using an intranasal DNA prime and intramuscular recombinant-vaccinia boost, that fusion-expressed CTB could serve as a potent adjuvant to enhance both systemic and mucosal T-cell responses. S. Dowall et al. describe early studies using in vitro models to investigate potential targets for immunomodulatory therapy during infection with the highly infectious Ebola virus. They showed binding of the phosphatidylserine-targeting antibody (bavituximab), which has already progressed through clinical trials (phases I-III) for treatment of tumours, to the surface of host cells infected with Ebola and also to purified Ebola virions. Assessing immune therapies for other indications has the potential to allow repurposing of products for treatment of biodefence agents, potentially saving significantly on research and development time and costs. In conclusion, this special issue has surveyed a diverse range of approaches to the development of biodefence vaccines and therapies and has explored the likely impact and application to the field of exciting new technological developments. Julia A. Tree E. Diane Williamson Caroline A. Rowland Louise M. Pitt

Summary A selection of previously identified protective Mycobacterium tuberculosis DNA vaccines were re-formulated as proteins and administered with a Th1-inducing adjuvant to help stimulate the relevant immune responses necessary for protection. All three candidate-vaccines conferred high levels of antigen-specific cellular and humoral responses, as indicated by lymphocyte proliferation and serum IgG levels. Protective efficacy was also assessed in comparison with the current vaccine, BCG (the ‘gold-standard' against which new vaccines are tested), and a saline (negative) control. One candidate (Rv1806-1807) induced protection in the guinea pig aerosol infection model 30 days post-challenge on the basis of reducing the bacterial burden of M. tuberculosis in the lungs.

Aims: To compare the inactivation of feline calicivirus (FCV) (a surrogate for Norovirus, NV) with the reduction of a bacterial water quality indicator (Escherichia coli), a human enteric virus (poliovirus) and a viral indicator (MS2, FRNA bacteriophage), following the disinfection of wastewaters. Methods and Results: Bench-scale disinfection experiments used wastewater (sterilized by gamma-irradiation) seeded with laboratory-cultured organisms. Seeded primary effluent was treated with different doses of applied free chlorine (8, 16 and 30 mg l−1). FCV and E. coli were easily inactivated by >4 log10, within 5 min with a dose of 30 mg l−1 of applied chlorine. Poliovirus was more resistant and a reduction of 2·85 log10 was seen after 30 min, MS2 was the most resistant organism (1 log10 inactivation). In further experiments seeded secondary effluent was treated with different doses of u.v. irradiation. To achieve a 4-log10 reduction of E. coli, FCV, poliovirus and MS2 doses of 5·32, 19·04, 27·51 and 62·50 mW s cm−2, respectively, were required. Conclusions: Feline calicivirus and E. coli seeded in primary wastewater were very susceptible to chlorination compared with poliovirus and MS2. In contrast, FCV seeded in secondary wastewater was more resistant to u.v. irradiation than E. coli but more sensitive than poliovirus and MS2. Significance and Impact of the Study: FRNA phage was more resistant to inactivation than all the viruses tested. This suggests FRNA phage would be a useful and conservative indicator of virus inactivation following disinfection of wastewaters with chlorination or u.v. irradiation.

SUMMARY Intranasal vaccination may offer practical benefits and better protection against respiratory infections, including tuberculosis. In this paper, we investigated the persistence of the Mycobacterium bovis-strain bacille Calmette–Guérin (BCG) Pasteur, lung granuloma formation and protection against pathogenic tuberculous challenge in mice. A pronounced BCG dose-dependent granulomatous infiltration of the lungs was observed following intranasal, but not after subcutaneous, vaccination. Corresponding doses of BCG, over a 100-fold range, imparted similar protection against H37Rv challenge when comparing the intranasal and subcutaneous vaccination routes. Interestingly, a BCG dose-dependent reduction of the H37Rv challenge infection was observed in the lungs, but not in the spleens, following both intranasal and subcutaneous vaccination. In the light of the observed concurrence between the extent of granuloma formation and the level of protection of the lungs, we conclude that intranasal vaccination leading to best protective efficacy needs to be balanced with an acceptable safety margin avoiding undue pathology in the lungs.

Wastewater disinfection is used in many countries for reducing fecal coliform levels in effluents. Disinfection is therefore frequently used to improve recreational bathing waters which do not comply with microbiological standards. It is unknown whether human enteric viruses (which are responsible for waterborne disease) are simultaneously inactivated alongside fecal coliforms. This laboratory study focused on the chlorination of primary treated effluent with three doses (8, 16, and 30 mg/liter) of free chlorine as sodium hypochlorite. Seeding experiments showed that inactivation (>5 log 10 units) of Escherichia coli and Enterococcus faecalis was rapid and complete but that there was poor inactivation (0.2 to 1.0 log 10 unit) of F + -specific RNA (FRNA) bacteriophage (MS2) (a potential virus indicator) at all three doses. However, seeded poliovirus was significantly more susceptible (2.8 log 10 units) to inactivation by chlorine than was the FRNA bacteriophage. To ensure that these results were not artifacts of the seeding process, comparisons were made between inactivation rates of laboratory-seeded organisms in sterilized sewage and inactivation rates of organisms occurring naturally in sewage. Multifactorial analysis of variance showed that there was no significant difference ( P > 0.05) between the inactivation rates for seeded and naturally occurring FRNA bacteriophage. However, laboratory-grown poliovirus was inactivated much more rapidly than were naturally occurring, indigenous enteroviruses ( P < 0.001). This may reflect differences in the way indigenous virus is presented to the disinfectant. Inactivation rates for indigenous enteroviruses were quite similar to those seen for FRNA bacteriophage at lower doses of chlorine. These results have significance for the effectiveness of chlorination as a sewage treatment process, particularly where virus contamination is of concern, and suggest that FRNA bacteriophage would be an appropriate indicator of such viral inactivation under field conditions.

Tuberculosis (TB) remains a major global public health problem. The only vaccine, BCG, gives variable protection, especially in adults, so several new vaccines are in clinical trials. There are no correlates of protective immunity to TB; therefore vaccines progress through lengthy and expensive pre-clinical assessments and human trials. Correlates of protection could act as early end-points during clinical trials, accelerating vaccine development and reducing costs. A genome-wide microarray was utilised to identify potential correlates of protection and biomarkers of disease induced post-BCG vaccination and post-Mycobacterium tuberculosis challenge in PPD-stimulated peripheral blood mononuclear cells from cynomolgus macaques where the outcome of infection was known. Gene expression post BCG-vaccination and post challenge was compared with gene expression when the animals were naive. Differentially expressed genes were identified using a moderated T test with Benjamini Hochberg multiple testing correction. After BCG vaccination and six weeks post-M. tuberculosis challenge, up-regulation of genes related to a Th1 and Th17 response was observed in disease controllers. At post-mortem, RT-PCR revealed an up-regulation of iron regulatory genes in animals that developed TB and down-regulation of these genes in disease controllers, indicating the ability to successfully withhold iron may be important in the control of TB disease. The induction of a balanced Th1 and Th17 response, together with expression of effector cytokines, such as IFNG, IL2, IL17, IL21 and IL22, could be used as correlates of a protective host response.

Different types of microcarriers were assessed for the large-scale culture of influenza virus in the Madin-Darby canine kidney (MDCK) cells. Both porous and solid carriers were examined. A higher titre of influenza A/PR8/34 virus was recovered from cultures using solid (1.3x10(9) PFU per ml) rather than porous carriers (4.0x10(8) PFU per ml). High titres of virus (1.0x10(9) PFU per ml) were also obtained from roller bottle cultures of MDCK cells and the traditional culture technique using embryonated hens' eggs (3.9x10(9) PFU per ml). We found that solid carriers composed of dextran with a positive charge are the most suitable carriers for the large-scale growth of influenza A virus in MDCK cells using serum-free media.

To support the licensure of a new and safer vaccine to protect people against smallpox, a monkeypox model of infection in cynomolgus macaques, which simulates smallpox in humans, was used to evaluate two vaccines, Acam2000 and Imvamune, for protection against disease. Animals vaccinated with a single immunization of Imvamune were not protected completely from severe and/or lethal infection, whereas those receiving either a prime and boost of Imvamune or a single immunization with Acam2000 were protected completely. Additional parameters, including clinical observations, radiographs, viral load in blood, throat swabs, and selected tissues, vaccinia virus-specific antibody responses, immunophenotyping, extracellular cytokine levels, and histopathology were assessed. There was no significant difference ( P > 0.05) between the levels of neutralizing antibody in animals vaccinated with a single immunization of Acam2000 (132 U/ml) and the prime-boost Imvamune regime (69 U/ml) prior to challenge with monkeypox virus. After challenge, there was evidence of viral excretion from the throats of 2 of 6 animals in the prime-boost Imvamune group, whereas there was no confirmation of excreted live virus in the Acam2000 group. This evaluation of different human smallpox vaccines in cynomolgus macaques helps to provide information about optimal vaccine strategies in the absence of human challenge studies.

Concern over the release of variola virus as an agent of bioterrorism remains high and a rapid vaccination regimen is desirable for use in the event of a confirmed release of virus. A single, high-dose (5×108 TCID50) of Bavarian Nordic's IMVAMUNE was tested in a Phase-II clinical trial, in humans, as a substitute for the standard (1×108 TCID50), using a 2-dose, 28-days apart regimen. Prior to this clinical trial taking place a Good Laboratory Practice, repeated high-dose, toxicology study was performed using IMVAMUNE, in New Zealand white rabbits and the results are reported here. Male and female rabbits were dosed twice, subcutaneously, with 5×108 TCID50 of IMVAMUNE (test) or saline (control), 7-days apart. The clinical condition, body-weight, food consumption, haematology, blood chemistry, immunogenicity, organ-weight, and macroscopic and microscopic pathology were investigated. Haematological investigations indicated changes within the white blood cell profile that were attributed to treatment with IMVAMUNE; these comprised slight increases in neutrophil and monocyte numbers, on study days 1-3 and a marginal increase in lymphocyte numbers on day 10. Macroscopic pathology revealed reddening at the sites of administration and thickened skin in IMVAMUNE, treated animals. After the second dose of IMVAMUNE 9/10 rabbits seroconverted, as detected by antibody ELISA on day 10, by day 21, 10/10 rabbits seroconverted. Treatment-related changes were not detected in other parameters. In conclusion, the subcutaneous injection of 2 high-doses of IMVAMUNE, to rabbits, was well tolerated producing only minor changes at the site of administration. Vaccinia-specific antibodies were raised in IMVAMUNE-vaccinated rabbits only.

Tuberculosis kills more than 1.5 million people per year, and standard treatment has remained unchanged for more than 30 years. Tuberculosis (TB) drives matrix metalloproteinase (MMP) activity to cause immunopathology. In advanced HIV infection, tissue destruction is reduced, but underlying mechanisms are poorly defined and no current antituberculous therapy reduces host tissue damage.To investigate MMP activity in patients with TB with and without HIV coinfection and to determine the potential of doxycycline to inhibit MMPs and decrease pathology.Concentrations of MMPs and cytokines were analyzed by Luminex array in a prospectively recruited cohort of patients. Modulation of MMP secretion and Mycobacterium tuberculosis growth by doxycycline was studied in primary human cells and TB-infected guinea pigs.HIV coinfection decreased MMP concentrations in induced sputum of patients with TB. MMPs correlated with clinical markers of tissue damage, further implicating dysregulated protease activity in TB-driven pathology. In contrast, cytokine concentrations were no different. Doxycycline, a licensed MMP inhibitor, suppressed TB-dependent MMP-1 and -9 secretion from primary human macrophages and epithelial cells by inhibiting promoter activation. In the guinea pig model, doxycycline reduced lung TB colony forming units after 8 weeks in a dose-dependent manner compared with untreated animals, and in vitro doxycycline inhibited mycobacterial proliferation.HIV coinfection in patients with TB reduces concentrations of immunopathogenic MMPs. Doxycycline decreases MMP activity in a cellular model and suppresses mycobacterial growth in vitro and in guinea pigs. Adjunctive doxycycline therapy may reduce morbidity and mortality in TB.

Immune responses in the guinea pig model are understudied because of a lack of commercial reagents. We have developed a custom-made guinea pig oligonucleotide microarray (81 spots) and have examined the gene expression profile of splenocytes restimulated in vitro from Mycobacterium bovis BCG-vaccinated and naive animals. Eleven genes were significantly ( P < 0.05) up-regulated following vaccination, indicating a Th1-type response. These results show that microarrays can be used to more fully define immune profiles of guinea pigs.

Immunization with plasmid DNA (pDNA) has the potential to overcome the difficulties of neonatal vaccination that may be required for protection against infection with respiratory syncytial virus (RSV); however, little is known about optimal delivery modalities. In this pilot study we compared mucosal delivery of pDNA encoding RSV F protein encapsulated in poly(DL-lactide-co-glycolide) with delivery of pDNA by gene-gun for the induction of immunity in mice. Intra-gastric or intra-nasal immunization with various doses of microparticles induced weak low levels of RSV-specific serum antibodies in a proportion of mice; in contrast, gene-gun vaccination led to protective immunity associated with a humoral response. Interestingly, RSV-specific antibody was detected in lung fragment cultures following intradermal vaccination with the gene-gun.