The ChAdOx1 vectored vaccine, AZD2816, induces strong immunogenicity against SARS-CoV-2 beta (B.1.351) and other variants of concern in preclinical studies.

BACKGROUND: There is an ongoing global effort to design, manufacture, and clinically assess vaccines against SARS-CoV-2. Over the course of the ongoing pandemic a number of new SARS-CoV-2 virus isolates or variants of concern (VoC) have been identified containing mutations in key proteins. METHODS: In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine (AZD2816) which expresses the spike protein of the Beta VoC (B.1.351). FINDINGS: We demonstrate that AZD2816 is immunogenic after a single dose. When AZD2816 is used as a booster dose in animals primed with a vaccine encoding the original spike protein (ChAdOx1 nCoV-19/ [AZD1222]), an increase in binding and neutralising antibodies against Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) is observed following each additional dose. In addition, a strong and polyfunctional T cell response was measured all booster regimens. INTERPRETATION: Real world data is demonstrating that one or more doses of licensed SARS-CoV-2 vaccines confer reduced protection against hospitalisation and deaths caused by divergent VoC, including Omicron. Our data support the ongoing clinical development and testing of booster vaccines to increase immunity against highly mutated VoC. FUNDING: This research was funded by AstraZeneca with supporting funds from MRC and BBSRC.

The SARS-CoV-2 monoclonal antibody combination, AZD7442, is protective in nonhuman primates and has an extended half-life in humans.

Despite the success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of coronavirus disease 2019 (COVID-19). Monoclonal antibodies (mAbs) against the viral spike protein have potential to both prevent and treat COVID-19 and reduce the risk of severe disease and death. Here, we describe AZD7442, a combination of two mAbs, AZD8895 (tixagevimab) and AZD1061 (cilgavimab), that simultaneously bind to distinct, nonoverlapping epitopes on the spike protein receptor binding domain to neutralize SARS-CoV-2. Initially isolated from individuals with prior SARS-CoV-2 infection, the two mAbs were designed to extend their half-lives and reduce effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry, and neutralize all tested SARS-CoV-2 variants of concern. In a nonhuman primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, whereas therapeutic administration accelerated virus clearance from the lung. In an ongoing phase 1 study in healthy participants (NCT04507256), a 300-mg intramuscular injection of AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers greater than 10-fold above those of convalescent serum for at least 3 months, which remained threefold above those of convalescent serum at 9 months after AZD7442 administration. About 1 to 2% of serum AZD7442 was detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentration suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19.

Genetic and structural basis for SARS-CoV-2 variant neutralization by a two-antibody cocktail.

Understanding the molecular basis for immune recognition of SARS-CoV-2 spike glycoprotein antigenic sites will inform the development of improved therapeutics. We determined the structures of two human monoclonal antibodies-AZD8895 and AZD1061-which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor-binding domain (RBD) of SARS-CoV-2 to define the genetic and structural basis of neutralization. AZD8895 forms an 'aromatic cage' at the heavy/light chain interface using germ line-encoded residues in complementarity-determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals. AZD1061 has an unusually long LCDR1; the HCDR3 makes interactions with the opposite face of the RBD from that of AZD8895. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the crucial binding residues of both antibodies and identified positions of concern with regards to virus escape from antibody-mediated neutralization. Both AZD8895 and AZD1061 have strong neutralizing activity against SARS-CoV-2 and variants of concern with antigenic substitutions in the RBD. We conclude that germ line-encoded antibody features enable recognition of the SARS-CoV-2 spike RBD and demonstrate the utility of the cocktail AZD7442 in neutralizing emerging variant viruses.

Severity of heterosubtypic influenza virus infection in ferrets is reduced by live attenuated influenza vaccine.

Live attenuated influenza vaccine (LAIV) is widely used to protect humans from seasonal influenza infection, particularly in children. In contrast to inactivated vaccines, the LAIV can induce both mucosal and cellular immune responses. Here we show that a single dose of monovalent H1N1pdm09-specific LAIV in the ferret model is fully protective against a subsequent wild-type H1N1pdm09 challenge, and furthermore reduces the severity of disease following challenge with a different influenza A subtype (H3N2). The reduced severity comprised reductions in weight loss and fever, as well as more rapid clearance of virus, compared to non-vaccinated H3N2-challenged ferrets. No H3N2-neutralizing antibodies were detected in vaccinated ferret sera. Rather, heterosubtypic protection correlated with interferon-gamma+ (IFN-γ+) T-cell responses measured in peripheral blood and in lung lymphocytes. The IFN-γ+ cells were cross-reactive to H3N2 virus even when obtained from vaccinated animals that had never been exposed to H3N2 virus. We believe this study provides compelling evidence that the LAIV can provide a significant reduction in infection and symptoms when challenged with heterosubtypic influenza strains not included in the LAIV, highlighting the importance of cross-reactive T-cells in the design of a universal influenza vaccine.

Defining the root cause of reduced H1N1 live attenuated influenza vaccine effectiveness: low viral fitness leads to inter-strain competition.

In the 2013-14 and 2015-16 influenza seasons, reduced vaccine effectiveness (VE) was observed for the H1N1 component of the FluMist quadrivalent live attenuated influenza vaccine (QLAIV) in the USA, leading to loss of Advisory Committee on Immunization Practices recommendation. Here we demonstrate in ferrets that 2015-16A/H1N1pdm09 vaccine strain A/Bolivia/559/2013 (A/BOL13) is outcompeted in trivalent (TLAIV) and QLAIV formulations, leading to reduced protection from wild-type challenge. While monovalent (MLAIV) A/BOL13 provided significant protection from wild-type virus shedding and fever at doses as low as 3.0 log10 fluorescent focus units (FFU), it failed to provide a similar level of protection in TLAIV or QLAIV formulation, even at a 6.0 log10 FFU dose. Conversely, clinically effective H1N1 strain A/New Caledonia/20/1999 provided significant protection in MLAIV, TLAIV, and QLAIV formulations. In conclusion, reduced A/BOL13 replicative fitness rendered it susceptible to inter-strain competition in QLAIV, contributing to its reduced VE in the 2015-16 season.

Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail.

The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an 'aromatic cage' at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1-4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.

Assessment of the benefits of seasonal influenza vaccination: Elements of a framework to interpret estimates of vaccine effectiveness and support robust decision-making and communication.

Systematic reviews and meta-analyses confirm that influenza vaccination reduces the risk of influenza illness by between about 40% and 60% in seasons when circulating influenza stains are well matched to vaccine strains. Influenza vaccine effectiveness (IVE) estimates, however, are often discordant and a source of confusion for decision makers. IVE assessments are increasingly publicized and are often used by policy makers to make decisions about the value of seasonal influenza vaccination. But there is limited guidance on how IVE should be interpreted or used to inform policy. There are several limitations to the use of IVE for decision-making: (a) IVE studies have methodological issues that often complicate the interpretation of their value; and (b) the full impact of vaccination will almost always be greater than the impact assessed by a point estimate of IVE in specific populations or settings. Understanding the strengths and weaknesses of study methodologies and the fundamental limitations of IVE estimates is important for the accuracy of interpretations and support of policy makers' decisions. Here, we review a comprehensive set of issues that need to be considered when interpreting IVE and determining the full benefits of influenza vaccination. We propose that published IVE values should be assessed using an evaluative framework that includes influenza-specific outcomes, types of VE study design, and confounders, among other factors. Better interpretation of IVE will improve the broader assessment of the value of influenza vaccination and ultimately optimize the public health benefits in seasonal influenza vaccination.

Replication of live attenuated influenza vaccine viruses in human nasal epithelial cells is associated with H1N1 vaccine effectiveness.

In the 2013-2014 and 2015-2016 influenza seasons, live attenuated influenza vaccine (LAIV) generated reduced vaccine effectiveness (VE) against circulating H1N1 strains. This reduced VE coincided with the introduction of pandemic 2009 H1N1 (A/H1N1pdm09) vaccine virus reassortants, in place of pre-2009 seasonal H1N1 strains. Here, we explored one specific hypothesis for reduced VE; decreased replicative fitness of A/H1N1pdm09 strains in humans. Two A/H1N1pdm09 strains with reduced VE, A/California/07/2009 (A/CA09) and A/Bolivia/559/2013 (A/BOL13), were compared to pre-2009 seasonal H1N1 strains, A/New Caledonia/20/1999 (A/NC99) and A/South Dakota/6/2007 (A/SD07). Initial results showed that A/H1N1pdm09 strains had reduced multi-cycle infectivity in Madin-Darby Canine Kidney (MDCK) cells, compared to their pre-2009 counterparts. The A/BOL13 viral titre was found to be 2.65 log10/mL lower when measured by multi-cycle 50% tissue culture infectious dose (TCID50) assay compared to single-cycle fluorescent focus assay (FFA). By contrast, clinically effective A/NC99 titres differed by only 0.54 log10/mL. In human alveolar (A549) cells, A/H1N1pdm09 strains replicated less than pre-2009 strains, with A/CA09 and A/BOL13 generating lower peak viral titres over 5 days. This phenotype was corroborated in physiologically relevant, primary human nasal epithelial cells (hNECs). Here, peak titres for pre-2009 strains A/NC99 and A/SD07 were 8.43 log10 TCID50/mL and 8.52 log10 TCID50/mL, respectively, versus 6.89 log10 TCID50/mL and 6.06 log10 TCID50/mL for A/H1N1pdm09 strains A/CA09 and A/BOL13. This confirmed a reduced ability of A/H1N1pdm09 strains to sustain replication in human respiratory cells. Using this information, H1N1 candidate A/Slovenia/2903/2015 (A/SLOV15) was characterised for replacement of A/BOL13 in the 2017/18 LAIV. A/SLOV15 produced comparable single and multi-cycle infectivity titres (Δ 0.16 log10/mL) and reached a peak titre 1.23 log10 TCID50/mL higher than that of A/BOL13 in hNEC cultures. Taken together, these data suggest a reduction in sustained multi-cycle replication in human cells as a plausible root cause for reduced A/H1N1pdm09 VE.

Development of hepatic pathology in GBV-B-infected red-bellied tamarins (Saguinus labiatus).

GB virus B (GBV-B) is a new world monkey-associated flavivirus used to model acute hepatitis C virus (HCV) infection. Critical for evaluation of antiviral or vaccine approaches is an understanding of the effect of HCV on the liver at different stages of infection. In the absence of longitudinal human tissue samples at defined time points, we have characterized changes in tamarins. As early as 2 weeks post-infection histological changes were noticeable, and these were established in all animals by 6 weeks. Despite high levels of liver-associated viral RNA, there was reversal of hepatic damage on clearance of peripheral virus though fibrosis was demonstrated in four tamarins. Notably, viral RNA burden in the liver dropped to near undetectable or background levels in all animals which underwent a second viral challenge, highlighting the efficacy of the immune response in removing foci of replication in the liver. These data add to the knowledge of GBV-B infection in New World primates which can offer attractive systems for the testing of prophylactic and therapeutic treatments and the evaluation of their utility in preventing or reversing liver pathology.

Haemagglutinin stability was not the primary cause of the reduced effectiveness of live attenuated influenza vaccine against A/H1N1pdm09 viruses in the 2013-2014 and 2015-2016 seasons.

During the 2013-2014 influenza season, the quadrivalent live attenuated influenza vaccine (QLAIV), had lower than expected vaccine effectiveness (VE) against circulating A/H1N1pdm09 viruses in the USA. The underlying reason proposed for this was that the A/H1N1pdm09 vaccine strain, A/California/07/2009 (A/CA09), had a thermally unstable haemagglutinin (HA) protein. Consequently, a new A/H1N1pdm09 candidate strain, A/Bolivia/559/2013 (A/BOL13), was developed for inclusion in the 2015-2016 QLAIV. A key parameter for selection of A/BOL13 was its more thermostable HA phenotype compared with A/CA09. During the 2015-2016 season, QLAIV containing A/BOL13 was found in some studies to have improved, but still with suboptimal, VE against circulating A/H1N1pdm09 viruses and was not recommended for use by the CDC in the US market in the 2016-2017 influenza season. This suggested that improved HA thermostability had not entirely resolved the reduced VE observed. One hypothesis for this was that, by improving thermostability, the A/BOL13 HA protein had been over-stabilised, compromising its activation at the low endosomal pH required for successful viral entry. Here we demonstrate that, while the A/BOL13 HA protein is more stable than that of A/CA09, its thermal and pH stability were comparable with historically efficacious LAIV strains, suggesting that the HA had not been over-stabilised. Furthermore, studies simulating potential heat exposure during distribution by exposing QLAIV nasal sprayers to 33 °C for 4 h showed that, while remaining within product specification, A/CA09 viral potency was statistically decreased after 12 weeks at 2-8 °C. These data suggest that although unfavourable HA protein stability may have contributed to the reduced VE of A/CA09 in 2013-2014, it was unlikely to have affected A/BOL13 in 2015-2016. We conclude that HA stability was not the primary cause of the reduced effectiveness of LAIV against A/H1N1pdm09 viruses in the 2013-2014 and 2015-2016 seasons.

Structured illumination microscopy combined with machine learning enables the high throughput analysis and classification of virus structure.

Optical super-resolution microscopy techniques enable high molecular specificity with high spatial resolution and constitute a set of powerful tools in the investigation of the structure of supramolecular assemblies such as viruses. Here, we report on a new methodology which combines Structured Illumination Microscopy (SIM) with machine learning algorithms to image and classify the structure of large populations of biopharmaceutical viruses with high resolution. The method offers information on virus morphology that can ultimately be linked with functional performance. We demonstrate the approach on viruses produced for oncolytic viriotherapy (Newcastle Disease Virus) and vaccine development (Influenza). This unique tool enables the rapid assessment of the quality of viral production with high throughput obviating the need for traditional batch testing methods which are complex and time consuming. We show that our method also works on non-purified samples from pooled harvest fluids directly from the production line.

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

Flaviviruses related to hepatitis C virus (HCV) in suitable animal models may provide further insight into the role that cellular immunity contributes to spontaneous clearance of HCV. We characterised changes in lymphocyte populations in tamarins with an acute GBV-B infection, a hepatitis virus of the flaviviridae. Major immune cell populations were monitored in peripheral and intra-hepatic lymphocytes at high viraemia or following a period when peripheral virus was no longer detected. Limited changes in major lymphocyte populations were apparent during high viraemia; however, the proportions of CD3(+) lymphocytes decreased and CD20(+) lymphocytes increased once peripheral viraemia became undetectable. Intrahepatic lymphocyte populations increased at both time points post-infection. Distinct expression patterns of PD-1, a marker of T-cell activation, were observed on peripheral and hepatic lymphocytes; notably there was elevated PD-1 expression on hepatic CD4(+) T-cells during high viraemia, suggesting an activated phenotype, which decreased following clearance of peripheral viraemia. At times when peripheral vRNA was not detected, suggesting viral clearance, we were able to readily detect GBV-B RNA in the liver, indicative of long-term virus replication. This study is the first description of changes in lymphocyte populations during GBV-B infection of tamarins and provides a foundation for more detailed investigations of the responses that contribute to the control of GBV-B infection.

Differences in respiratory syncytial virus and influenza infection in a house-dust-mite-induced asthma mouse model: consequences for steroid sensitivity.

A significant number of clinical asthma exacerbations are triggered by viral infection. We aimed to characterize the effect of virus infection in an HDM (house dust mite) mouse model of asthma and assess the effect of oral corticosteroids. HDM alone significantly increased eosinophils, lymphocytes, neutrophils, macrophages and a number of cytokines in BAL (bronchoalveolar lavage), all of which were sensitive to treatment with prednisolone (with the exception of neutrophils). Virus infection also induced cell infiltration and cytokines. RSV (respiratory syncytial virus) infection in HDM-treated animals further increased all cell types in BAL (except eosinophils, which declined), but induced no further increase in HDM-elicited cytokines. However, while HDM-elicited TNF-α (tumour necrosis factor-α), IFN-γ (interferon-γ), IL (interleukin)-2, IL-5 and IL-10 were sensitive to prednisolone treatment, concomitant infection with RSV blocked the sensitivity towards steroid. In contrast, influenza infection in HDM- challenged animals resulted in increased BAL lymphocytes, neutrophils, IFN-γ, IL-1ß, IL-4, IL-5, IL-10 and IL-12, but all were attenuated by prednisolone treatment. HDM also increased eNO (exhaled NO), which was further increased by concomitant virus infection. This increase was only partially attenuated by prednisolone. RSV infection alone increased BAL mucin. However, BAL mucin was increased in HDM animals with virus infection. Chronic HDM challenge in mice elicits a broad inflammatory response that shares many characteristics with clinical asthma. Concomitant influenza or RSV infection elicits differing inflammatory profiles that differ in their sensitivity towards steroids. This model may be suitable for the assessment of novel pharmacological interventions for asthmatic exacerbation.

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.

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.

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.

Therapy with TLR7 agonists induces lymphopenia: correlating pharmacology to mechanism in a mouse model.

BACKGROUND AND OBJECTIVE: Synthetic TLR7 agonists have been proposed as oral replacements for interferonα (IFNα) therapy in the treatment of hepatitis C virus infection. However, adverse effects, such as lymphopenia and cardiovascular irregularities, have been observed in the clinical following treatment with TLR7 agonists. We wished to understand and characterise the relationship between TLR7 agonism and adverse effects. METHODS: We compared responses to two prototypic TLR7 agonists (Resiquimod: R-848; and PF-04878691) in a mouse model and compared the responses to treatment with IFNα. We measured clinically relevant adverse effects such as lymphopenia and cardiovascular irregularities and related them to plasma drug levels and clinically relevant efficacy biomarkers such as the pro-inflammatory cytokine IP-10, 2'5'OAS and TLR7 receptor expression. RESULTS: By 2 h post dose all agents had induced a dose-dependent transient lymphopenia. IFNα increased heart rate immediately following dosing, persisting for 5 h, whilst PF-04878691 induced significant reductions in blood pressure. Lymphopenia co-incided with maximum plasma drug levels, raised levels of IP-10 and the auto-induction of TLR7 expression in the blood and lymph nodes. Peak levels of 2'5'OAS occurred at 24 h post-dose and only at doses which also induced lymphopenia. CONCLUSIONS: We conclude that systemic delivery of TLR7 agonists or IFNα induces similar exaggerated pharmacology, consistent with there being a narrow therapeutic window between efficacy and safety. This clinically validated mouse model will help to investigate whether more potent agonists or optimised dosing schedules, will be successful strategies for targeting TLR7 in patients.

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.