Helen Bright

A recombinant trimeric surfactant protein D carbohydrate recognition domain inhibits respiratory syncytial virus infection in vitro and in vivo

The pulmonary collectin, lung surfactant protein D (SP‐D), plays a role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP‐D. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G protein from RSV (human, A2 strain) and both native and recombinant human SP‐D. The effect of recombinant trimeric SP‐D lectin domains (rSP‐D) on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep‐2C cells and in vivo infections in BALB / c mice. These studies show that full‐length and recombinant SP‐D bind to RSV G protein in a concentration‐dependent manner. Both EDTA and mannan inhibited binding of full‐length SP‐D. These results indicate that binding occurs via the carbohydrate recognition domain of the SP‐D. The recombinant SP‐D inhibited RSV infectivity in cell culture in a dose‐dependent manner, giving 100 % inhibition of replication. Intranasal administration of recombinant SP‐D to RSV‐infected mice inhibited replication of the virus in the lungs, reducing levels of lung virus by 80 %. These results suggest that SP‐D plays a major role in clearing RSV from the lungs.

Antiviral applications of Toll-like receptor agonists

In the past, antiviral research has focused mainly on viral targets. As the search for effective and differentiated antiviral therapies continues, cellular targets are becoming more common, bringing with them a variety of challenges and concerns. Toll-like receptors (TLRs) provide a unique mechanism to induce an antiviral state in the host. In this review we introduce TLRs as targets for the pharmaceutical industry, including how they signal and thereby induce an antiviral state through the production of type I interferons. We examine how TLRs are being therapeutically targeted and discuss several clinically precedented agents for which efficacy and safety data are available. We describe some of the chemistries that have been applied to both small molecule and large molecule leads to tune agonist potency, and offer a differentiated safety profile through targeting certain compartments such as the gut or the lung, thereby limiting systemic drug exposure and affecting systemic cytokine levels. The application of low-dose agonists of TLRs as vaccine adjuvants or immunoprotective agents is also presented. Some of the challenges presented by this approach are then discussed, including viral evasion strategies and mechanism-linked inflammatory cytokine induction.

Single and Combination Herpes Simplex Virus Type 2 Glycoprotein Vaccines Adjuvanted with CpG Oligodeoxynucleotides or Monophosphoryl Lipid A Exhibit Differential Immunity That Is Not Correlated to Protection in Animal Models

ABSTRACT Despite several attempts to develop an effective prophylactic vaccine for HSV-2, all have failed to show efficacy in the clinic. The most recent of these failures was the GlaxoSmithKline (GSK) subunit vaccine based on the glycoprotein gD with the adjuvant monophosphoryl lipid A (MPL). In a phase 3 clinical trial, this vaccine failed to protect from HSV-2 disease, even though good neutralizing antibody responses were elicited. We aimed to develop a superior, novel HSV-2 vaccine containing either gD or gB alone or in combination, together with the potent adjuvant CpG oligodeoxynucleotides (CPG). The immunogenic properties of these vaccines were compared in mice. We show that gB/CPG/alum elicited a neutralizing antibody response similar to that elicited by gD/CPG/alum vaccine but a significantly greater gamma interferon (IFN-γ) T cell response. Furthermore, the combined gB-gD/CPG/alum vaccine elicited significantly greater neutralizing antibody and T cell responses than gD/MPL/alum. The efficacies of these candidate vaccines were compared in the mouse and guinea pig disease models, including a novel male guinea pig genital disease model. These studies demonstrated that increased immune response did not correlate to improved protection. First, despite a lower IFN-γ T cell response, the gD/CPG/alum vaccine was more effective than gB/CPG/alum in mice. Furthermore, the gB-gD/CPG/alum vaccine was no more effective than gD/MPL/alum in mice or male guinea pigs. We conclude that difficulties in correlating immune responses to efficacy in animal models will act as a deterrent to researchers attempting to develop effective HSV vaccines.

The efficacy of HSV-2 vaccines based on gD and gB is enhanced by the addition of ICP27.

DNA vaccines expressing HSV-2 gD, gB, ICP27, VP22 and VP13/14 were shown to be immunogenic in mice; gD and gB elicited neutralising antibody, and all five antigens induced T cell responses measured by IFNγ ELISPOT. In murine HSV-2 challenge studies, gD and gB provided moderate to high levels of protection while ICP27 provided a lower level of protection depending on the model (intravaginal or intranasal) and the challenge dose. Combining vaccines expressing gB or gD with vaccines expressing ICP27 provided greater protection than any antigen alone. We conclude that the addition of ICP27 to enhance the anti-viral T cell response can improve the efficacy of gD- and gB-based vaccines.

Use of a highly sensitive strand-specific quantitative PCR to identify abortive replication in the mouse model of respiratory syncytial virus disease

BackgroundThe BALB/c mouse is commonly used to study RSV infection and disease. However, despite the many advantages of this well-characterised model, the inoculum is large, viral replication is restricted and only a very small amount of virus can be recovered from infected animals. A key question in this model is the fate of the administered virus. Is replication really being measured or is the model measuring the survival of the virus over time? To answer these questions we developed a highly sensitive strand-specific quantitative PCR (QPCR) able to accurately quantify the amount of RSV replication in the BALB/c mouse lung, allowing characterisation of RSV negative and positive strand RNA dynamics.ResultsIn the mouse lung, no increase in RSV genome was seen above the background of the original inoculum whilst only a limited transient increase (< 1 log) in positive strand, replicative intermediate (RI) RNA occurred. This RNA did however persist at detectable levels for 59 days post infection. As expected, ribavirin therapy reduced levels of infectious virus and RI RNA in the mouse lung. However, whilst Palivizumab therapy was also able to reduce levels of infectious virus, it failed to prevent production of intracellular RI RNA. A comparison of RSV RNA kinetics in human (A549) and mouse (KLN205) cell lines demonstrated that RSV replication was also severely delayed and impaired in vitro in the mouse cells.ConclusionsThis is the first time that such a sensitive strand-specific QPCR technique has been to the RSV mouse system. We have accurately quantified the restricted and abortive nature of RSV replication in the mouse. Further in vitro studies in human and mouse cells suggest this restricted replication is due at least in part to species-specific host cell-viral interactions.

Non-benzimidazole containing inhibitors of respiratory syncytial virus

Several non-benzimidazole containing inhibitors of respiratory syncytial virus are described. Core template modification, analysis of antiviral activity, physicochemistry and optimisation of properties led to the thiazole-imidazole 13, that showed a good potency and pharmacokinetic profile in the rat.

CD8+ T Lymphocyte Epitopes From The Herpes Simplex Virus Type 2 ICP27, VP22 and VP13/14 Proteins To Facilitate Vaccine Design And Characterization

CD8+ T cells have the potential to control HSV-2 infection. However, limited information has been available on CD8+ T cell epitopes or the functionality of antigen specific T cells during infection or following immunization with experimental vaccines. Peptide panels from HSV-2 proteins ICP27, VP22 and VP13/14 were selected from in silico predictions of binding to human HLA-A*0201 and mouse H-2Kd, Ld and Dd molecules. Nine previously uncharacterized CD8+ T cell epitopes were identified from HSV-2 infected BALB/c mice. HSV-2 specific peptide sequences stabilized HLA-A*02 surface expression with intermediate or high affinity binding. Peptide specific CD8+ human T cell lines from peripheral blood lymphocytes were generated from a HLA-A*02+ donor. High frequencies of peptide specific CD8+ T cell responses were elicited in mice by DNA vaccination with ICP27, VP22 and VP13/14, as demonstrated by CD107a mobilization. Vaccine driven T cell responses displayed a more focused immune response than those induced by viral infection. Furthermore, vaccination with ICP27 reduced viral shedding and reduced the clinical impact of disease. In conclusion, this study describes novel HSV-2 epitopes eliciting strong CD8+ T cell responses that may facilitate epitope based vaccine design and aid immunomonitoring of antigen specific T cell frequencies in preclinical and clinical settings.

Pathologic and virologic characterization of neuroinvasion by HSV-2 in a mouse encephalitis model

Herpes simplex virus type 2 (HSV-2), a ubiquitous human pathogenassociated with genital infections, is neurotropic. It establishes latent infections in local dorsal root ganglia from which it reactivates causing recurrent lesions and frequent episodes of viral shedding. Herpes simplex virus type 2 can also be transmitted from mother to child during birth, causing major neonatal complications including encephalitis. Animal models of HSV-2 genital infection are well described and used for testing of therapies; little is known about animal models of HSV-2-induced encephalitis. We analyzed the pathologic and immunohistochemical features of the nasal rostrum and brain tissueand correlated them with viral distribution in a mouse model of HSV-2 encephalitis induced by intranasal infection and examined viral replication in the brain tissue using quantitative polymerase chain reaction and traditional plaque assay. Our results suggest that the primary route for HSV-2 neuroinvasion after intranasal infection is via thetrigeminal pathway, ultimately leading to infection of the brainstem and meningoencephalitis.

Changes in immune cell populations in the periphery and liver of GBV-B-infected and convalescent tamarins (Saguinus labiatus)

Highlights • GBV-B infection of tamarins is a valuable model for acute HCV infection.• We observed distinct expression patterns of PD-1, a marker of T-cell activation, on peripheral and hepatic lymphocytes.• Differential PD-1 expression is coincident with reduction in peripheral GBV-B.• Liver-associated viral RNA in the absence of peripheral viraemia indicates maintenance of occult infection.

Mouse model of HDM induced airway inflammation + influenza or RSV. Effects of steroid

Influenza A virus is one of the most common infectious pathogens. The disease that ensues is particularly serious in certain at risk groups within the population; such as individuals with chronic respiratory and cardiovascular diseases. Asthma is an example of a chronic inflammatory disease which is exacerbated by the virus, and infection frequently leads to hospitalization of asthmatic patients. The role of respiratory viruses in development of severe clinical asthma, in particular, is poorly understood. Pfizer has supported the European Union-led IMI UBIOPRED pre-competitive collaboration between academic and pharma partners which aims to better understand the clinical disease, and in doing so, developed pre-clinical models of both severe asthma and asthma exacerbation. We established and characterised RSV and Influenza A infection in the mouse house dust mite (HDM) model. We have examined the bronchoalveolar fluid (bal) cell and lung cytokine responses as well as observing the health of the animals for a 14 day period following exposure to varying virus doses. Groups of mice were infected with 5 x virus doses and virus replication and inflammatory responses monitored at days 1, 3, 7 and 14 post infection. Bronchoalveolar lavage (BAL) and lungs were collected from all mice. BAL was processed for cell counts and tissue was processed for cytokine and pathology. Additionally, mice were observed daily for onset of clinical symptoms and weight loss. We have demonstrated two unique viral exacerbation profiles and demonstrated that influenza can generate an exacerbative profile in conjunction with HDM challenge.