Mediation of antitumor activity by AZD4820 oncolytic vaccinia virus encoding IL-12.

Oncolytic viruses are engineered to selectively kill tumor cells and have demonstrated promising results in early-phase clinical trials. To further modulate the innate and adaptive immune system, we generated AZD4820, a vaccinia virus engineered to express interleukin-12 (IL-12), a potent cytokine involved in the activation of natural killer (NK) and T cells and the reprogramming of the tumor immune microenvironment. Testing in cultured human tumor cell lines demonstrated broad in vitro oncolytic activity and IL-12 transgene expression. A surrogate virus expressing murine IL-12 demonstrated antitumor activity in both MC38 and CT26 mouse syngeneic tumor models that responded poorly to immune checkpoint inhibition. In both models, AZD4820 significantly upregulated interferon-gamma (IFN-γ) relative to control mice treated with oncolytic vaccinia virus (VACV)-luciferase. In the CT26 study, 6 of 10 mice had a complete response after treatment with AZD4820 murine surrogate, whereas control VACV-luciferase-treated mice had 0 of 10 complete responders. AZD4820 treatment combined with anti-PD-L1 blocking antibody augmented tumor-specific T cell immunity relative to monotherapies. These findings suggest that vaccinia virus delivery of IL-12, combined with immune checkpoint blockade, elicits antitumor immunity in tumors that respond poorly to immune checkpoint inhibitors.

The genomic evolutionary dynamics and global circulation patterns of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection in young children and the second leading cause of infant death worldwide. While global circulation has been extensively studied for respiratory viruses such as seasonal influenza, and more recently also in great detail for SARS-CoV-2, a lack of global multi-annual sampling of complete RSV genomes limits our understanding of RSV molecular epidemiology. Here, we capitalise on the genomic surveillance by the INFORM-RSV study and apply phylodynamic approaches to uncover how selection and neutral epidemiological processes shape RSV diversity. Using complete viral genome sequences, we show similar patterns of site-specific diversifying selection among RSVA and RSVB and recover the imprint of non-neutral epidemic processes on their genealogies. Using a phylogeographic approach, we provide evidence for air travel governing the global patterns of RSVA and RSVB spread, which results in a considerable degree of phylogenetic mixing across countries. Our findings highlight the potential of systematic global RSV genomic surveillance for transforming our understanding of global RSV spread.

Seropersistence of SII-ChAdOx1 nCoV-19 (COVID-19 vaccine): 6-month follow-up of a randomized, controlled, observer-blind, phase 2/3 immuno-bridging study in Indian adults.

AZD1222 (ChAdOx1 nCoV-19) is a replication-deficient adenoviral vectored coronavirus disease-19 (COVID-19) vaccine that is manufactured as SII-ChAdOx1 nCoV-19 by the Serum Institute of India Pvt Ltd following technology transfer from Oxford University/AstraZeneca. The non-inferiority of SII-ChAdOx1 nCoV-19 with AZD1222 was previously demonstrated in an observer-blind, phase 2/3 immuno-bridging study (trial registration: CTRI/2020/08/027170). In this analysis of immunogenicity and safety data 6 months post first vaccination (Day 180), 1,601 participants were randomized 3:1 to SII-ChAdOx1 nCoV-19 or AZD1222 (immunogenicity/reactogenicity cohort n = 401) and 3:1 to SII-ChAdOx1 nCoV-19 or placebo (safety cohort n = 1,200). Immunogenicity was measured by anti-severe acute respiratory syndrome coronavirus 2 spike (anti-S) binding immunoglobulin G and neutralizing antibody (nAb) titers. A decline in anti-S titers was observed in both vaccine groups, albeit with a greater decline in SII-ChAdOx1 nCoV-19 vaccinees (geometric mean titer [GMT] ratio [95% confidence interval (CI) of SII-ChAdOx1 nCoV-19 to AZD1222]: 0.60 [0.41-0.87]). Consistent similar decreases in nAb titers were observed between vaccine groups (GMT ratio [95% CI]: 0.88 [0.44-1.73]). No cases of severe COVID-19 were reported following vaccination, while one case was observed in the placebo group. No causally related serious adverse events were reported through 180 days. No thromboembolic or autoimmune adverse events of special interest were reported. Collectively, these data illustrate that SII-ChAdOx1 nCoV-19 maintained a high level of immunogenicity 6 months post-vaccination. SII-ChAdOx1 nCoV-19 was safe and well tolerated.

Analysis of SARS-CoV-2 Emergent Variants Following AZD7442 (Tixagevimab/Cilgavimab) for Early Outpatient Treatment of COVID-19 (TACKLE Trial).

INTRODUCTION: AZD7442 (tixagevimab/cilgavimab) comprises neutralising monoclonal antibodies (mAbs) that bind to distinct non-overlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Viral evolution during mAb therapy can select for variants with reduced neutralisation susceptibility. We examined treatment-emergent SARS-CoV-2 variants during TACKLE (NCT04723394), a phase 3 study of AZD7442 for early outpatient treatment of coronavirus disease 2019 (COVID-19). METHODS: Non-hospitalised adults with mild-to-moderate COVID-19 were randomised and dosed ≤ 7 days from symptom onset with AZD7442 (n = 452) or placebo (n = 451). Next-generation sequencing of the spike gene was performed on SARS-CoV-2 reverse-transcription polymerase chain reaction-positive nasopharyngeal swabs at baseline and study days 3, 6, and 15 post dosing. SARS-CoV-2 lineages were assigned using spike nucleotide sequences. Amino acid substitutions were analysed at allele fractions (AF; % of sequence reads represented by substitution) ≥ 25% and 3% to 25%. In vitro susceptibility to tixagevimab, cilgavimab, and AZD7442 was evaluated for all identified treatment-emergent variants using a pseudotyped microneutralisation assay. RESULTS: Longitudinal spike sequences were available for 461 participants (AZD7442, n = 235; placebo, n = 226) and showed that treatment-emergent variants at any time were rare, with 5 (2.1%) AZD7442 participants presenting ≥ 1 substitution in tixagevimab/cilgavimab binding sites at AF ≥ 25%. At AF 3% to 25%, treatment-emergent variants were observed in 15 (6.4%) AZD7442 and 12 (5.3%) placebo participants. All treatment-emergent variants showed in vitro susceptibility to AZD7442. CONCLUSION: These data indicate that AZD7442 creates a high genetic barrier for resistance and is a feasible option for COVID-19 treatment.

Immunogenicity and safety of AZD2816, a beta (B.1.351) variant COVID-19 vaccine, and AZD1222 (ChAdOx1 nCoV-19) as third-dose boosters for previously vaccinated adults: a multicentre, randomised, partly double-blinded, phase 2/3 non-inferiority immunobridging study in the UK and Poland.

BACKGROUND: This study aimed to evaluate AZD2816, a variant-updated COVID-19 vaccine expressing the full-length SARS-CoV-2 beta (B.1.351) variant spike protein that is otherwise similar to AZD1222 (ChAdOx1 nCoV-19), and AZD1222 as third-dose boosters. METHODS: This phase 2/3, partly double-blinded, randomised, active-controlled study was done at 19 sites in the UK and four in Poland. Adult participants who had received a two-dose AZD1222 or mRNA vaccine primary series were randomly assigned by means of an Interactive Response Technology-Randomisation and Trial Supply Management system (1:1 within each primary-series cohort, stratified by age, sex, and comorbidities) to receive AZD1222 or AZD2816 (intramuscular injection; 5 × 1010 viral particles). Participants, investigators, and all sponsor staff members involved in study conduct were masked to randomisation. AZD1222 and AZD2816 doses were prepared by unmasked study staff members. The primary objectives were to evaluate safety and humoral immunogenicity (non-inferiority of day-29 pseudovirus neutralising antibody geometric mean titre [GMT] against ancestral SARS-CoV-2: AZD1222 booster vs AZD1222 primary series [historical controls]; margin 0·67; SARS-CoV-2-seronegative participants). This study is registered with ClinicalTrials.gov, NCT04973449, and is completed. FINDINGS: Between June 27 and Sept 30, 2021, 1394 participants of the 1741 screened were randomly assigned to AZD1222 or AZD2816 following an AZD1222 (n=373, n=377) or mRNA vaccine (n=322, n=322) primary series. In SARS-CoV-2-seronegative participants receiving AZD1222 or AZD2816, 78% and 80% (AZD1222 primary series) and 90% and 93%, respectively (mRNA vaccine primary series) reported solicited adverse events to the end of day 8; 2%, 2%, 1%, and 1% had serious adverse events and 12%, 12%, 10%, and 11% had adverse events of special interest, respectively, to the end of day 180. The primary immunogenicity non-inferiority endpoint was met: day-29 neutralising antibody GMT ratios (ancestral SARS-CoV-2) were 1·02 (95% CI 0·90-1·14) and 3·47 (3·09-3·89) with AZD1222 booster versus historical controls (AZD1222 and mRNA vaccine primary series, respectively). Responses against beta were greater with AZD2816 versus AZD1222 (GMT ratios, AZD1222, mRNA vaccine primary series 1·84 [1·63-2·08], 2·22 [1·99-2·47]). INTERPRETATION: Both boosters were well tolerated, with immunogenicity against ancestral SARS-CoV-2 similar to AZD1222 primary-series vaccination. AZD2816 gave greater immune responses against beta versus AZD1222. FUNDING: AstraZeneca.

Fc-mediated functions of nirsevimab complement direct respiratory syncytial virus neutralization but are not required for optimal prophylactic protection.

Introduction: Nirsevimab is an extended half-life (M252Y/S254T/T256E [YTE]-modified) monoclonal antibody to the pre-fusion conformation of the respiratory syncytial virus (RSV) Fusion protein, with established efficacy in preventing RSV-associated lower respiratory tract infection in infants for the duration of a typical RSV season. Previous studies suggest that nirsevimab confers protection via direct virus neutralization. Here we use preclinical models to explore whether fragment crystallizable (Fc)-mediated effector functions contribute to nirsevimab-mediated protection. Methods: Nirsevimab, MEDI8897* (i.e., nirsevimab without the YTE modification), and MEDI8897*-TM (i.e., MEDI8897* without Fc effector functions) binding to Fc γ receptors (FcγRs) was evaluated using surface plasmon resonance. Antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent complement deposition (ADCD), and antibody-dependent cellular cytotoxicity (ADCC) were assessed through in vitro and ex vivo serological analyses. A cotton rat challenge study was performed with MEDI8897* and MEDI8897*-TM to explore whether Fc effector functions contribute to protection from RSV. Results: Nirsevimab and MEDI8897* exhibited binding to a range of FcγRs, with expected reductions in FcγR binding affinities observed for MEDI8897*-TM. Nirsevimab exhibited in vitro ADNP, ADCP, ADCD, and ADCC activity above background levels, and similar ADNP, ADCP, and ADCD activity to palivizumab. Nirsevimab administration increased ex vivo ADNP, ADCP, and ADCD activity in participant serum from the MELODY study (NCT03979313). However, ADCC levels remained similar between nirsevimab and placebo. MEDI8897* and MEDI8897*-TM exhibited similar dose-dependent reduction in lung and nasal turbinate RSV titers in the cotton rat model. Conclusion: Nirsevimab possesses Fc effector activity comparable with the current standard of care, palivizumab. However, despite possessing the capacity for Fc effector activity, data from RSV challenge experiments illustrate that nirsevimab-mediated protection is primarily dependent on direct virus neutralization.

Molecular and phenotypic characteristics of RSV infections in infants during two nirsevimab randomized clinical trials.

Nirsevimab is a monoclonal antibody that binds to the respiratory syncytial virus (RSV) fusion protein. During the Phase 2b (NCT02878330) and MELODY (NCT03979313) clinical trials, infants received one dose of nirsevimab or placebo before their first RSV season. In this pre-specified analysis, isolates from RSV infections were subtyped, sequenced and analyzed for nirsevimab binding site substitutions; subsequently, recombinant RSVs were engineered for microneutralization susceptibility testing. Here we show that the frequency of infections caused by subtypes A and B is similar across and within the two trials. In addition, RSV A had one and RSV B had 10 fusion protein substitutions occurring at >5% frequency. Notably, RSV B binding site substitutions were rare, except for the highly prevalent I206M:Q209R, which increases nirsevimab susceptibility; RSV B isolates from two participants had binding site substitutions that reduce nirsevimab susceptibility. Overall, >99% of isolates from the Phase 2b and MELODY trials retained susceptibility to nirsevimab.

Breakthrough SARS-CoV-2 Infections in the PROVENT Prevention Trial Were Not Associated With AZD7442 (Tixagevimab/Cilgavimab) Resistant Variants.

BACKGROUND: We report spike protein-based lineage and AZD7442 (tixagevimab/cilgavimab) neutralizing activity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants identified from breakthrough infections in the PROVENT preexposure prophylaxis trial. METHODS: Variants identified from PROVENT participants with reverse-transcription polymerase chain reaction-positive symptomatic illness were phenotypically assessed to determine neutralization susceptibility of variant-specific pseudotyped virus-like particles. RESULTS: At completion of 6 months' follow-up, no AZD7442-resistant variants were observed in breakthrough coronavirus disease 2019 (COVID-19) cases. SARS-CoV-2 neutralizing antibody titers were similar in breakthrough and nonbreakthrough cases. CONCLUSIONS: Symptomatic COVID-19 breakthrough cases in PROVENT were not due to resistance-associated substitutions in AZD7442 binding sites or lack of AZD7442 exposure. CLINICAL TRIALS REGISTRATION: NCT04625725.

One year safety and immunogenicity of AZD1222 (ChAdOx1 nCoV-19): Final analysis of a randomized, placebo-controlled phase 1/2 trial in Japan.

BACKGROUND: Long duration trial data for two-dose COVID-19 vaccines primary series' are uncommon due to unblinding and additional doses. We report one-year follow-up results from a phase 1/2 trial of AZD1222 (ChAdOx1 nCoV-19) in Japan. METHODS: Adults (n = 256) seronegative for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) were stratified by age, 18-55 (n = 128), 56-69 (n = 86) and ≥70-year-old (n = 42), and randomized 3:1 to AZD1222 or placebo. Safety, immunogenicity, and exploratory efficacy data were collected until study Day 365. RESULTS: Safety was consistent with previous reports. In AZD1222 vaccinees, humoral responses against SARS-CoV-2 steadily declined over time. By Day 365, anti-SARS-CoV-2 spike-binding (spike) and receptor-binding domain (RBD) mean antibody titers remained above Day 15 levels and pseudovirus neutralizing antibodies were undetectable in many participants. CONCLUSIONS: AZD1222 is immunogenic and well tolerated in Japanese adults. Expected waning in anti-SARS-CoV-2 humoral responses was observed; spike and RBD antibody titers remained elevated. (ClinicalTrials.gov: NCT04568031).

Molecular Characterization of AZD7442 (Tixagevimab-Cilgavimab) Neutralization of SARS-CoV-2 Omicron Subvariants.

Therapeutic anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (MAbs) provide immunosuppressed and vulnerable populations with prophylactic and treatment interventions against coronavirus disease 2019 (COVID-19). AZD7442 (tixagevimab-cilgavimab) is a combination of extended-half-life neutralizing MAbs that bind to distinct epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The Omicron variant of concern carries mutations at >35 positions in the spike protein and has undergone further genetic diversification since its emergence in November 2021. Here, we characterize the in vitro neutralization activity of AZD7442 toward major viral subvariants circulating worldwide during the first 9 months of the Omicron wave. BA.2 and its derived subvariants showed the highest susceptibility to AZD7442, while BA.1 and BA.1.1 showed a lower susceptibility. BA.4/BA.5 had a susceptibility level intermediate between BA.1 and BA.2. Mutagenesis of parental Omicron subvariant spike proteins was performed to establish a molecular model to describe the underlying determinants of neutralization by AZD7442 and its component MAbs. The concurrent mutation of residues at positions 446 and 493, located in the tixagevimab and cilgavimab binding sites, was sufficient to enhance in vitro susceptibility of BA.1 to AZD7442 and its component MAbs to levels similar to the Wuhan-Hu-1+D614G virus. AZD7442 maintained neutralization activity against all Omicron subvariants tested up to and including BA.5. The evolving nature of the SARS-CoV-2 pandemic warrants continuing real-time molecular surveillance and assessment of in vitro activity of MAbs used in prophylaxis against and the treatment of COVID-19. IMPORTANCE MAbs are key therapeutic options for COVID-19 prophylaxis and treatment in immunosuppressed and vulnerable populations. Due to the emergence of SARS-CoV-2 variants, including Omicron, it is vital to ensure that neutralization is maintained for MAb-based interventions. We studied the in vitro neutralization of AZD7442 (tixagevimab-cilgavimab), a cocktail of two long-acting MAbs targeting the SARS-CoV-2 spike protein, toward Omicron subvariants circulating from November 2021 to July 2022. AZD7442 neutralized major Omicron subvariants up to and including BA.5. The mechanism of action responsible for the lower in vitro susceptibility of BA.1 to AZD7442 was investigated using in vitro mutagenesis and molecular modeling. A combination of mutations at two spike protein positions, namely, 446 and 493, was sufficient to enhance BA.1 susceptibility to AZD7442 to levels similar to the Wuhan-Hu-1+D614G ancestral virus. The evolving nature of the SARS-CoV-2 pandemic warrants continuing real-time global molecular surveillance and mechanistic studies of therapeutic MAbs for COVID-19.

Immune correlates analysis of a phase 3 trial of the AZD1222 (ChAdOx1 nCoV-19) vaccine.

In the phase 3 trial of the AZD1222 (ChAdOx1 nCoV-19) vaccine conducted in the U.S., Chile, and Peru, anti-spike binding IgG concentration (spike IgG) and pseudovirus 50% neutralizing antibody titer (nAb ID50) measured four weeks after two doses were assessed as correlates of risk and protection against PCR-confirmed symptomatic SARS-CoV-2 infection (COVID-19). These analyses of SARS-CoV-2 negative participants were based on case-cohort sampling of vaccine recipients (33 COVID-19 cases by 4 months post dose two, 463 non-cases). The adjusted hazard ratio of COVID-19 was 0.32 (95% CI: 0.14, 0.76) per 10-fold increase in spike IgG concentration and 0.28 (0.10, 0.77) per 10-fold increase in nAb ID50 titer. At nAb ID50 below the limit of detection (< 2.612 IU50/ml), 10, 100, and 270 IU50/ml, vaccine efficacy was -5.8% (-651%, 75.6%), 64.9% (56.4%, 86.9%), 90.0% (55.8%, 97.6%) and 94.2% (69.4%, 99.1%). These findings provide further evidence towards defining an immune marker correlate of protection to help guide regulatory/approval decisions for COVID-19 vaccines.

Nirsevimab binding-site conservation in respiratory syncytial virus fusion glycoprotein worldwide between 1956 and 2021: an analysis of observational study sequencing data.

BACKGROUND: Nirsevimab is an extended half-life monoclonal antibody to the respiratory syncytial virus (RSV) fusion protein that has been developed to protect infants for an entire RSV season. Previous studies have shown that the nirsevimab binding site is highly conserved. However, investigations of the geotemporal evolution of potential escape variants in recent (ie, 2015-2021) RSV seasons have been minimal. Here, we examine prospective RSV surveillance data to assess the geotemporal prevalence of RSV A and B, and functionally characterise the effect of the nirsevimab binding-site substitutions identified between 2015 and 2021. METHODS: We assessed the geotemporal prevalence of RSV A and B and nirsevimab binding-site conservation between 2015 and 2021 from three prospective RSV molecular surveillance studies (the US-based OUTSMART-RSV, the global INFORM-RSV, and a pilot study in South Africa). Nirsevimab binding-site substitutions were assessed in an RSV microneutralisation susceptibility assay. We contextualised our findings by assessing fusion-protein sequence diversity from 1956 to 2021 relative to other respiratory-virus envelope glycoproteins using RSV fusion protein sequences published in NCBI GenBank. FINDINGS: We identified 5675 RSV A and RSV B fusion protein sequences (2875 RSV A and 2800 RSV B) from the three surveillance studies (2015-2021). Nearly all (25 [100%] of 25 positions of RSV A fusion proteins and 22 [88%] of 25 positions of RSV B fusion proteins) amino acids within the nirsevimab binding site remained highly conserved between 2015 and 2021. A highly prevalent (ie, >40·0% of all sequences) nirsevimab binding-site Ile206Met:Gln209Arg RSV B polymorphism arose between 2016 and 2021. Nirsevimab neutralised a diverse set of recombinant RSV viruses, including new variants containing binding-site substitutions. RSV B variants with reduced susceptibility to nirsevimab neutralisation were detected at low frequencies (ie, prevalence <1·0%) between 2015 and 2021. We used 3626 RSV fusion-protein sequences published in NCBI GenBank between 1956 and 2021 (2024 RSV and 1602 RSV B) to show that the RSV fusion protein had lower genetic diversity than influenza haemagglutinin and SARS-CoV-2 spike proteins. INTERPRETATION: The nirsevimab binding site was highly conserved between 1956 and 2021. Nirsevimab escape variants were rare and have not increased over time. FUNDING: AstraZeneca and Sanofi.

AZD1222-induced nasal antibody responses are shaped by prior SARS-CoV-2 infection and correlate with virologic outcomes in breakthrough infection.

The nasal mucosa is an important initial site of host defense against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, intramuscularly administered vaccines typically do not achieve high antibody titers in the nasal mucosa. We measure anti-SARS-CoV-2 spike immunoglobulin G (IgG) and IgA in nasal epithelial lining fluid (NELF) following intramuscular vaccination of 3,058 participants from the immunogenicity substudy of a phase 3, double-blind, placebo-controlled study of AZD1222 vaccination (ClinicalTrials.gov: NCT04516746). IgG is detected in NELF collected 14 days following the first AZD1222 vaccination. IgG levels increase with a second vaccination and exceed pre-existing levels in baseline-SARS-CoV-2-seropositive participants. Nasal IgG responses are durable and display strong correlations with serum IgG, suggesting serum-to-NELF transudation. AZD1222 induces short-lived increases to pre-existing nasal IgA levels in baseline-seropositive vaccinees. Vaccinees display a robust recall IgG response upon breakthrough infection, with overall magnitudes unaffected by time between vaccination and illness. Mucosal responses correlate with reduced viral loads and shorter durations of viral shedding in saliva.

AZD7442 (Tixagevimab/Cilgavimab) for Post-Exposure Prophylaxis of Symptomatic Coronavirus Disease 2019.

BACKGROUND: This phase 3 trial assessed AZD7442 (tixagevimab/cilgavimab) for post-exposure prophylaxis against symptomatic coronavirus disease 2019 (COVID-19). METHODS: Adults without prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or COVID-19 vaccination were enrolled within 8 days of exposure to a SARS-CoV-2-infected individual and randomized 2:1 to a single 300-mg AZD7442 dose (one 1.5-mL intramuscular injection each of tixagevimab and cilgavimab) or placebo. Primary end points were safety and first post-dose SARS-CoV-2 reverse-transcription polymerase chain reaction (RT-PCR)-positive symptomatic COVID-19 event before day 183. RESULTS: A total of 1121 participants were randomized and dosed (AZD7442, n = 749; placebo, n = 372). Median (range) follow-up was 49 (5-115) and 48 (20-113) days for AZD7442 and placebo, respectively. Adverse events occurred in 162 of 749 (21.6%) and 111 of 372 (29.8%) participants with AZD7442 and placebo, respectively, mostly mild/moderate. RT-PCR-positive symptomatic COVID-19 occurred in 23 of 749 (3.1%) and 17 of 372 (4.6%) AZD7442- and placebo-treated participants, respectively (relative risk reduction, 33.3%; 95% confidence interval [CI], -25.9 to 64.7; P = .21). In predefined subgroup analyses of 1073 (96%) participants who were SARS-CoV-2 RT-PCR-negative (n = 974, 87%) or missing an RT-PCR result (n = 99, 9%) at baseline, AZD7442 reduced RT-PCR-positive symptomatic COVID-19 by 73.2% (95% CI, 27.1 to 90.1) vs placebo. CONCLUSIONS: This study did not meet the primary efficacy end point of post-exposure prevention of symptomatic COVID-19. However, analysis of participants who were SARS-CoV-2 RT-PCR-negative or missing an RT-PCR result at baseline support a role for AZD7442 in preventing symptomatic COVID-19. Clinical Trials Registration. NCT04625972.

Tolerability and immunogenicity of an intranasally-administered adenovirus-vectored COVID-19 vaccine: An open-label partially-randomised ascending dose phase I trial.

BACKGROUND: Intranasal vaccination may induce protective local and systemic immune responses against respiratory pathogens. A number of intranasal SARS-CoV-2 vaccine candidates have achieved protection in pre-clinical challenge models, including ChAdOx1 nCoV-19 (AZD1222, University of Oxford / AstraZeneca). METHODS: We performed a single-centre open-label Phase I clinical trial of intranasal vaccination with ChAdOx1 nCoV-19 in healthy adults, using the existing formulation produced for intramuscular administration. Thirty SARS-CoV-2 vaccine-naïve participants were allocated to receive 5 × 109 viral particles (VP, n=6), 2 × 1010 VP (n=12), or 5 × 1010 VP (n=12). Fourteen received second intranasal doses 28 days later. A further 12 received non-study intramuscular mRNA SARS-CoV-2 vaccination between study days 22 and 46. To investigate intranasal ChAdOx1 nCoV-19 as a booster, six participants who had previously received two intramuscular doses of ChAdOx1 nCoV-19 and six who had received two intramuscular doses of BNT162b2 (Pfizer / BioNTech) were given a single intranasal dose of 5 × 1010 VP of ChAdOx1 nCoV-19. Objectives were to assess safety (primary) and mucosal antibody responses (secondary). FINDINGS: Reactogenicity was mild or moderate. Antigen-specific mucosal antibody responses to intranasal vaccination were detectable in a minority of participants, rarely exceeding levels seen after SARS-CoV-2 infection. Systemic responses to intranasal vaccination were typically weaker than after intramuscular vaccination with ChAdOx1 nCoV-19. Antigen-specific mucosal antibody was detectable in participants who received an intramuscular mRNA vaccine after intranasal vaccination. Seven participants developed symptomatic SARS-CoV-2 infection. INTERPRETATION: This formulation of intranasal ChAdOx1 nCoV-19 showed an acceptable tolerability profile but induced neither a consistent mucosal antibody response nor a strong systemic response. FUNDING: AstraZeneca.

Durability of protection and immunogenicity of AZD1222 (ChAdOx1 nCoV-19) COVID-19 vaccine over 6 months.

BackgroundWe report updated safety, efficacy, and immunogenicity of AZD1222 (ChAdOx1 nCoV-19) from an ongoing phase 3 trial.MethodsAdults at increased risk of SARS-CoV-2 infection were randomized (2:1), stratified by age, to receive 2 doses of AZD1222 or placebo. The primary efficacy end point was confirmed SARS-CoV-2 reverse-transcriptase PCR-positive (RT-PCR-positive) symptomatic COVID-19 at 15 or more days after a second dose in baseline SARS-CoV-2-seronegative participants. The 21,634 and 10,816 participants were randomized to AZD1222 and placebo, respectively.FindingsData cutoff for this analysis was July 30, 2021; median follow-up from second dose was 78 and 71 days for the double-blind period (censoring at unblinding or nonstudy COVID-19 vaccination) and 201 and 82 days for the period to nonstudy COVID-19 vaccination (regardless of unblinding) in the AZD1222 and placebo groups, respectively. For the primary efficacy end point in the double-blind period (141 and 184 events; incidence rates: 39.2 and 118.8 per 1,000 person years), vaccine efficacy was 67.0% (P < 0.001). In the period to nonstudy COVID-19 vaccination, incidence of events remained consistently low and stable through 6 months in the AZD1222 group; for the primary efficacy end point (328 and 219 events; incidence rates: 36.4, 108.4) and severe/critical disease (5 and 13 events; incidence rates: 0.6, 6.4), respective vaccine efficacy estimates were 65.1% and 92.1%. AZD1222 elicited humoral immune responses over time, with waning at day 180. No emergent safety issues were seen.ConclusionAZD1222 is safe and well tolerated, demonstrating durable protection and immunogenicity with median follow-up (AZD1222 group) of 6 months.Trial registrationClinicalTrials.gov NCT04516746.FundingAstraZeneca; US government.

Qualification of a Biolayer Interferometry Assay to Support AZD7442 Resistance Monitoring.

AZD7442, a combination of two long-acting monoclonal antibodies (tixagevimab [AZD8895] and cilgavimab [AZD1061]), has been authorized for the prevention and treatment of coronavirus disease 2019 (COVID-19). The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires methods capable of quickly characterizing resistance to AZD7442. To support AZD7442 resistance monitoring, a biolayer interferometry (BLI) assay was developed to screen the binding of tixagevimab and cilgavimab to SARS-CoV-2 spike proteins to reduce the number of viral variants for neutralization susceptibility verification. Six spike variants were chosen to assess the assay's performance: four with decreased affinity for tixagevimab (F486S:D614G and F486W:D614G proteins) or cilgavimab (S494L:D614G and K444R:D614G proteins) and two reference proteins (wild-type HexaPro and D614G protein). Equilibrium dissociation constant (KD) values from each spike protein were used to determine shifts in binding affinity. The assay's precision, range, linearity, and limits of quantitation were established. Qualification acceptance criteria determined whether the assay was fit for purpose. By bypassing protein purification, the BLI assay provided increased screening throughput. Although limited correlation between pseudotype neutralization and BLI data (50% inhibitory concentration versus KD) was observed for full immunoglobulins (IgGs), the correlations for antibody fragments (Fabs) were stronger and reflected a better comparison of antibody binding kinetics with neutralization potency. Therefore, despite strong assay performance characteristics, the use of full IgGs limited the screening utility of the assay; however, the Fab approach warrants further exploration as a rapid, high-throughput variant-screening method for future resistance-monitoring programs. IMPORTANCE SARS-CoV-2 variants harbor multiple substitutions in their spike trimers, potentially leading to breakthrough infections and clinical resistance to immune therapies. For this reason, a BLI assay was developed and qualified to evaluate the reliability of screening SARS-CoV-2 spike trimer variants against anti-SARS-CoV-2 monoclonal antibodies (MAbs) tixagevimab and cilgavimab, the components of AZD7442, prior to in vitro pseudovirus neutralization susceptibility verification testing. The assay bypasses protein purification with rapid assessment of the binding affinity of each MAb for each recombinant protein, potentially providing an efficient preliminary selection step, thus allowing a reduced testing burden in the more technically complex viral neutralization assays. Despite precise and specific measures, an avidity effect associated with MAb binding to the trimer confounded correlation with neutralization potency, negating the assay's utility as a surrogate for neutralizing antibody potency. Improved correlation with Fabs suggests that assay optimization could overcome any avidity limitation, warranting further exploration to support future resistance-monitoring programs.

Validation and performance of a multiplex serology assay to quantify antibody responses following SARS-CoV-2 infection or vaccination.

Objectives: Robust, quantitative serology assays are required to accurately measure antibody levels following vaccination and natural infection. We present validation of a quantitative, multiplex, SARS-CoV-2, electrochemiluminescent (ECL) serology assay; show correlation with two established SARS-CoV-2 immunoassays; and present calibration results for two SARS-CoV-2 reference standards. Methods: Precision, dilutional linearity, ruggedness, analytical sensitivity and specificity were evaluated. Clinical sensitivity and specificity were assessed using serum from prepandemic and SARS-CoV-2 polymerase chain reaction (PCR)-positive patient samples. Assay concordance to the established Roche Elecsys® Anti-SARS-CoV-2 immunoassay and a live-virus microneutralisation (MN) assay was evaluated. Results: Standard curves demonstrated the assay can quantify SARS-CoV-2 antibody levels over a broad range. Assay precision (10.2-15.1% variability), dilutional linearity (≤ 1.16-fold bias per 10-fold increase in dilution), ruggedness (0.89-1.18 overall fold difference), relative accuracy (107-118%) and robust selectivity (102-104%) were demonstrated. Analytical sensitivity was 7, 13 and 7 arbitrary units mL-1 for SARS-CoV-2 spike (S), receptor-binding domain (RBD) and nucleocapsid (N) antigens, respectively. For all antigens, analytical specificity was > 90% and clinical specificity was 99.0%. Clinical sensitivities for S, RBD and N antigens were 100%, 98.8% and 84.9%, respectively. Comparison with the Elecsys® immunoassay showed ≥ 87.7% agreement and linear correlation (Pearson r of 0.85, P < 0.0001) relative to the MN assay. Conversion factors for the WHO International Standard and Meso Scale Discovery® Reference Standard are presented. Conclusions: The multiplex SARS-CoV-2 ECL serology assay is suitable for efficient, reproducible measurement of antibodies to SARS-CoV-2 antigens in human sera, supporting its use in clinical trials and sero-epidemiology studies.