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1.
researchsquare; 2022.
Preprint in English | PREPRINT-RESEARCHSQUARE | ID: ppzbmed-10.21203.rs.3.rs-2244796.v1

ABSTRACT

SARS-CoV-2 continues to circulate in the human population necessitating regular booster immunization for its long-term control. Ideally, vaccines should ideally not only protect against symptomatic disease, but also prevent transmission via asymptomatic shedding and cover existing and future variants of the virus. This may ultimately only be possible through induction of potent and long-lasting immune responses in the nasopharyngeal tract, the initial entry site of SARS-CoV-2. To this end, we have designed a vaccine based on recombinantly expressed receptor binding domain (RBD) of SARS-CoV-2, fused to the C-terminus of C. perfringens enterotoxin (cCPE), which is known to target Claudin-4, a matrix molecule highly expressed on mucosal microfold (M) cells of the nasal and bronchial-associated lymphoid tissues. To further enhance immune responses, the vaccine was adjuvanted with a novel toll-like receptor 3/RIG-I agonist (Riboxxim™), consisting of synthetic short double stranded RNA. Intranasal prime-boost immunization of mice induced robust mucosal and systemic anti-SARS-CoV-2 neutralizing antibody responses against SARS-CoV-2 strains Wuhan-Hu-1, and several variants (B.1.351/beta, B.1.1.7/alpha, B.1.617.2/delta), as well as systemic T-cell responses. A combination vaccine with M-cell targeted recombinant HA1 from an H1N1 G4 influenza strain also induced mucosal and systemic antibodies against influenza. Taken together, the data show that development of an intranasal SARS-CoV-2 vaccine based on recombinant RBD adjuvanted with a TLR3 agonist is feasible, also as a combination vaccine against influenza.

2.
biorxiv; 2022.
Preprint in English | bioRxiv | ID: ppzbmed-10.1101.2022.05.21.492554

ABSTRACT

The Omicron lineage of SARS-CoV-2, first described in November 2021, spread rapidly to become globally dominant and has split into a number of sub-lineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africa's Gauteng region uncovered two new sub-lineages, BA.4 and BA.5 which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences and, although closely related to BA.2, contain further mutations in the receptor binding domain of spike. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by serum from triple AstraZeneca or Pfizer vaccinated individuals compared to BA.1 and BA.2. Furthermore, using serum from BA.1 vaccine breakthrough infections there are likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections.

3.
researchsquare; 2022.
Preprint in English | PREPRINT-RESEARCHSQUARE | ID: ppzbmed-10.21203.rs.3.rs-995273.v1

ABSTRACT

Of the coronaviruses that have caused zoonotic spill overs in past two decades, the diverse group of beta-coronaviruses (β-CoVs) represent the greatest threats. Towards achieving broad vaccine protection from these viruses, vaccines composed of multiple antigens, each capable of eliciting broad neutralising responses across a subgroup will be required. Utilising a novel platform for selecting immune optimized, structurally engineered antigens capable of eliciting protective responses across a group of related viruses, we demonstrate proof-concept against the greater sarbecoviruses sub-genus with a single antigen structure. From an array of phylogenetically informed antigen structures displaying different broad neutralising epitopes, synthetic genes expressing these were selected based on broad immune responses in BALB/C mice. Improved protection against the Delta variant was further observed in K18-hACE2 mice on boosting with the lead designs of mice primed by an approved COVID-19 vaccine. Immunogenicity of the lead vaccine antigen and breadth of neutralisation against the SARS-CoV, SARS-CoV-2, WIV16, and RaTG13 was confirmed in guinea pigs using needleless intradermal immunisation. Immunogenicity was further confirmed in rabbits with GMP manufactured DNA immunogen. Notably, given the increasing number of mutations acquired by SARS-CoV-2 variants of concern (VOCs), the rabbit sera were tested for the capacity to neutralise VOCs - Beta, Gamma, Delta, as well as the most diverse Omicron variant. The consistent neutralising ability of the vaccine sera against the emerging VOCs validate broad specificity of the vaccine design. Here, we demonstrate proof-of-concept of this Digitally Immune Optimised, Selected vaccine (DIOSvax) antigen pipeline for the in vivo selection of single nucleic acid-based immunogens. Such gene-based antigens can be readily delivered alone or in combination, and seamlessly scaled with vaccine delivery modalities such as viral vector or mRNA-based vaccines.

4.
biorxiv; 2021.
Preprint in English | bioRxiv | ID: ppzbmed-10.1101.2021.12.03.471045

ABSTRACT

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced. Omicron contains a total of 30 substitutions plus deletions and an insertion in Spike, far more than any previously reported variant. The mutations include those previously identified by In-vitro evolution to contribute to high-affinity binding to ACE2, including mutations Q498R and N501Y critical in forming additional interactions in the interface. Together with increased charge complementarity between the RBD and ACE2, these substantially increase affinity and potentially virus transmissibility through increased syncytia formation. Further mutations promote immune evasion. We have studied the binding of a large panel of potent monoclonal antibodies generated from early pandemic or Beta infected cases. Mutations in Omicron will likely compromise the binding of many of these and additionally, the binding of antibodies under commercial development, however residual binding should provide protection from severe disease.

5.
medrxiv; 2021.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2021.09.29.21264328

ABSTRACT

The virus SARS-CoV-2, responsible for the global COVID-19 pandemic, spread rapidly around the world causing high morbidity and mortality because humans have no pre-existing immunity. However, there are four known, endemic seasonal coronaviruses in humans (HCoVs) and whether antibodies for these HCoVs play a role in severity of COVID-19 disease has generated a lot of interest. Of these seasonal viruses NL63 is of particular interest as it uses the same cell entry receptor as SARS-CoV-2.We use functional, neutralising assays to investigate cross reactive antibodies and their relationship with COVID-19 severity. We analysed neutralisation of SARS-CoV-2, NL63, HKU1, and 229E in 38 COVID-19 patients and 62 healthcare workers, and a further 182 samples to specifically study the relationship between SARS-CoV-2 and NL63.We found that although HCoV neutralisation was very common there was little evidence that these antibodies neutralised SARS-CoV-2. Despite no evidence in cross neutralisation, levels of NL63 neutralisating antibodies become elevated after exposure to SARS-CoV-2 through infection or following vaccination.

6.
biorxiv; 2021.
Preprint in English | bioRxiv | ID: ppzbmed-10.1101.2021.08.17.456606

ABSTRACT

RaTG13 is a close relative of SARS-CoV-2, the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, sharing 96% sequence similarity at the genome-wide level. The spike receptor binding domain (RBD) of RaTG13 contains a large number of amino acid substitutions when compared to SARS-CoV-2, likely impacting affinity for the ACE2 receptor. Antigenic differences between the viruses are less well understood, especially whether RaTG13 spike can be efficiently neutralised by antibodies generated from infection with, or vaccination against, SARS-CoV-2. Using RaTG13 and SARS-CoV-2 pseudotypes we compared neutralisation using convalescent sera from previously infected patients as well as vaccinated healthcare workers. Surprisingly, our results revealed that RaTG13 was more efficiently neutralised than SARS-CoV-2. In addition, neutralisation assays using spike chimeras and mutants harbouring single amino acid substitutions within the RBD demonstrated that both spike proteins can tolerate multiple changes without dramatically reducing how efficiently they are neutralised. Moreover, introducing the 484K mutation into RaTG13 resulted in increased neutralisation, in contrast to the same mutation in SARS-CoV-2 (E484K). This is despite E484K having a well-documented role in immune evasion in variants of concern (VOC) such as B.1.351 (Beta). These results indicate that the immune-escape mutations found in SARS-CoV-2 VOCs might be driven by strong antibody pressures, and that the future spill-over of RaTG13 and/or related sarbecoviruses could be mitigated using current SARS-CoV-2-based vaccination strategies.

8.
medrxiv; 2021.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2021.05.21.21257572

ABSTRACT

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD, and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study, was to identify biomarkers of humoral immunity that could be used as candidate CoP in internationally accepted unitage. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (INU) for virus neutralisation assays or International Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG / IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD / S binding assays. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

9.
medrxiv; 2021.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2021.05.04.21256571

ABSTRACT

It is unclear whether prior endemic coronavirus infections affect COVID-19 severity. Here, we show that in cases of fatal COVID-19, antibody responses to the SARS-COV-2 spike are directed against epitopes shared with endemic beta-coronaviruses in the S2 subunit of the SARS-CoV-2 spike protein. This immune response is associated with the compromised production of a de novo SARS-CoV-2 spike response among individuals with fatal COVID-19 outcomes.

10.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.12.05.20241927

ABSTRACT

SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2, and amino acid variation in Spike is increasingly appreciated. Given both vaccines and therapeutics are designed around Wuhan-1 Spike, this raises the theoretical possibility of virus escape, particularly in immunocompromised individuals where prolonged viral replication occurs. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences by both short and long read technologies over 23 time points spanning 101 days. Although little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days, N501Y in Spike was transiently detected at day 55 and V157L in RdRp emerged. However, following convalescent plasma we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and{Delta} H69/{Delta}V70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro, the Spike escape double mutant bearing{Delta} H69/{Delta}V70 and D796H conferred decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility, but incurred an infectivity defect. The{Delta} H69/{Delta}V70 single mutant had two-fold higher infectivity compared to wild type and appeared to compensate for the reduced infectivity of D796H. Consistent with the observed mutations being outside the RBD, monoclonal antibodies targeting the RBD were not impacted by either or both mutations, but a non RBD binding monoclonal antibody was less potent against{Delta} H69/{Delta}V70 and the double mutant. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with reduced susceptibility to neutralising antibodies.

11.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.08.22.20176834

ABSTRACT

COVID-19 continues to cause a pandemic, having infected more than 20 million people globally. Successful elimination of the SARS-CoV-2 virus will require an effective vaccine. However, the immune correlates of infection are currently poorly understood. While neutralizing antibodies are believed to be essential for protection against infection, the contribution of the neutralizing antibody response to resolution of SARS-CoV-2 infection has not yet been defined. In this study the antibody responses to the SARS-CoV-2 spike protein and nucleocapsid proteins were investigated in a UK patient cohort, using optimised immunoassays and a retrovirus-based pseudotype entry assay. It was discovered that in severe COVID-19 infections an early antibody response to both antigens was associated with improved prognosis of infection. While not all SARS-CoV-2-reactive sera were found to possess neutralizing antibodies, neutralizing potency of sera was found to be greater in patients who went on to resolve infection, compared with those that died from COVID-19. Furthermore, viral genetic variation in spike protein was found to influence the production of neutralizing antibodies. Infection with the recently described spike protein variant 614G produced higher levels of neutralizing antibodies when compared to viruses possessing the 614D variant. These findings support the assertion that vaccines targeting generation of neutralizing antibodies may be useful at limiting SARS-CoV-2 infection. Assessment of the antibody responses to SARS-CoV-2 at time of diagnosis will be a useful addition to the diagnostic toolkit, enabling stratification of clinical intervention for severe COVID-19 disease.

12.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.07.09.20148429

ABSTRACT

Antibody (Ab) responses to SARS-CoV-2 can be detected in most infected individuals 10-15 days following the onset of COVID-19 symptoms. However, due to the recent emergence of this virus in the human population it is not yet known how long these Ab responses will be maintained or whether they will provide protection from re-infection. Using sequential serum samples collected up to 94 days post onset of symptoms (POS) from 65 RT-qPCR confirmed SARS-CoV-2-infected individuals, we show seroconversion in >95% of cases and neutralizing antibody (nAb) responses when sampled beyond 8 days POS. We demonstrate that the magnitude of the nAb response is dependent upon the disease severity, but this does not affect the kinetics of the nAb response. Declining nAb titres were observed during the follow up period. Whilst some individuals with high peak ID50 (>10,000) maintained titres >1,000 at >60 days POS, some with lower peak ID50 had titres approaching baseline within the follow up period. A similar decline in nAb titres was also observed in a cohort of seropositive healthcare workers from Guys and St Thomas Hospitals. We suggest that this transient nAb response is a feature shared by both a SARS-CoV-2 infection that causes low disease severity and the circulating seasonal coronaviruses that are associated with common colds. This study has important implications when considering widespread serological testing, Ab protection against re-infection with SARS-CoV-2 and the durability of vaccine protection.

13.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.06.16.20133157

ABSTRACT

BackgroundRapid COVID-19 diagnosis in hospital is essential for patient management and identification of infectious patients to limit the potential for nosocomial transmission. The diagnosis of infection is complicated by 30-50% of COVID-19 hospital admissions with nose/throat swabs testing negative for SARS-CoV-2 nucleic acid, frequently after the first week of illness when SARS-CoV-2 antibody responses become detectable. We assessed the diagnostic accuracy of combined rapid antibody point of care (POC) and nucleic acid assays for suspected COVID-19 disease in the emergency department. MethodsWe developed (i) an in vitro neutralization assay using a lentivirus expressing a genome encoding luciferase and pseudotyped with spike (S) protein and (ii) an ELISA test to detect IgG antibodies to nucleocapsid (N) and S proteins from SARS-CoV-2. We tested two lateral flow rapid fingerprick tests with bands for IgG and IgM. We then prospectively recruited participants with suspected moderate to severe COVID-19 and tested for SARS-CoV-2 nucleic acid in a combined nasal/throat swab using the standard laboratory RT-PCR and a validated rapid POC nucleic acid amplification (NAAT) test. Additionally, serum collected at admission was retrospectively tested by in vitro neutralisation, ELISA and the candidate POC antibody tests. We evaluated the performance of the individual and combined rapid POC diagnostic tests against a composite reference standard of neutralisation and standard laboratory based RT-PCR. Results45 participants had specimens tested for nucleic acid in nose/throat swabs as well as stored sera for antibodies. Using the composite reference standard, prevalence of COVID-19 disease was 53.3% (24/45). Median age was 73.5 (IQR 54.0-86.5) years in those with COVID-19 disease by our reference standard and 63.0 (IQR 41.0-72.0) years in those without disease. The overall detection rate by rapid NAAT was 79.2% (95CI 57.8-92.9%), decreasing from 100% (95% CI 65.3-98.6%) in days 1-4 to 50.0% (95% CI 11.8-88.2) for days 9-28 post symptom onset. Correct identification of COVID-19 with combined rapid POC diagnostic tests was 100% (95CI 85.8-100%) with a false positive rate of 5.3-14.3%, driven by POC LFA antibody tests. ConclusionsCombined POC tests have the potential to transform our management of COVID-19, including inflammatory manifestations later in disease where nucleic acid test results are negative. A rapid combined approach will also aid recruitment into clinical trials and in prescribing therapeutics, particularly where potentially harmful immune modulators (including steroids) are used.

14.
researchsquare; 2020.
Preprint in English | PREPRINT-RESEARCHSQUARE | ID: ppzbmed-10.21203.rs.3.rs-33080.v2

ABSTRACT

This work aims to assess the feasibility of performing COVID-19 RNA tests in hospitals and communities experiencing SARS-CoV-2 virus outbreaks, to ultimately provide recommendations for hospitals with so-called fever clinics. In China, these specialized clinics within a hospital specifically receive outpatients with fever symptoms. A team with expertise in the Exposure Analysis and Critical Control Points (EACCP) framework identified potential infection routes during the testing for SARS-CoV-2, then constructed and tested flow diagrams, which were confirmed under actual conditions, demonstrating the feasibility to carry out in hospitals with fever clinics. The team determined critical control points to mitigate the exposure risks at each control point. The sampling and inactivation steps of clinical samples in fever clinics appeared to be associated with particularly high-risk levels of exposure to SARS-CoV-2. Moderate exposure levels were associated with storage and transportation of samples for inactivation; Low-risk levels associated with the transportation, storage, and detection steps after inactivation. To minimize infection risks for personnel, we proposed optimized processes to carry out SARS-CoV-2 RNA tests in hospitals with fever clinics in China. The high risk of SARS-CoV-2 exposure during procedures preceding testing is the sampling and biological inactivation; Simultaneously, full personal protective equipment and BSL2 laboratories in fever clinics or mobile BSL2 laboratories could reduce the risk. Implementing the EACCP framework could facilitate rapid responses to outbreaks of emerging infectious diseases.

15.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.05.20.20091694

ABSTRACT

Introduction. The lack of approved specific therapeutic agents to treat COVID-19 associated with SARS coronavirus 2 (SARS-CoV-2) infection has led to the rapid implementation and/or randomised controlled trials of convalescent plasma therapy (CPT) in many countries including the UK. Effective CPT is likely to require high titres of neutralising antibody levels in convalescent donations. Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS-CoV-2 proteins in scalable assays will be crucial for the success of large-scale collection and use of convalescent plasma. We assessed whether neutralising antibody titres correlated with reactivity in a range of ELISA assays targeting the spike (S) protein, the main target for human immune response. Methods. Blood samples were collected from 52 individuals with a previous laboratory confirmed SARS-CoV-2 infection at least 28 days after symptom resolution. These were assayed for SARS-CoV-2 neutralising antibodies by microneutralisation and pseudotype assays, and for antibodies by four different ELISAs. ROC analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high neutralising antibody levels suitable for clinical use of convalescent plasma. Results. All samples contained SARS-CoV-2 antibodies, whereas neutralising antibody titres of greater than 1:20 were detected in 43 samples (83% of those tested) and >1:100 in 22 samples (42%). The best correlations were observed with EUROimmun IgG ELISA S/CO reactivity (Spearman Rho correlation co-efficient 0.88; p<0.001). Based on ROC analysis, EUROimmun would detect 60% of samples with titres of >1:100 with 100% specificity using a reactivity index of 9.1 (13/22). Discussion. Robust associations between virus neutralising antibody titres and reactivity in several ELISA-based antibody tests demonstrate their possible utility for scaled-up production of convalescent plasma containing potentially therapeutic levels of anti-SARS-CoV-2 neutralising antibodies.

16.
medrxiv; 2020.
Preprint in English | medRxiv | ID: ppzbmed-10.1101.2020.04.13.20060467

ABSTRACT

BackgroundThe progression and geographical distribution of SARS coronavirus 2 (SARS-CoV-2) infection in the UK and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of blood donors in Scotland between the 17th of March and the 18th of May to detect neutralising antibodies to SARS-CoV-2 as a marker of past infection and epidemic progression. AimTo determine if sera from blood bank donors can be used to track the emergence and progression of the SARS-CoV-2 epidemic. MethodsA pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study group comprised samples from 3,500 blood donors collected in Scotland between the 17th of March and 19th of May, 2020. Controls were collected from 100 donors in Scotland during 2019. ResultsAll samples collected on the 17th March, 2020 (n=500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in 6/500 donors from the 23th-26th of March. The number of samples containing neutralising antibodies did not significantly rise after the 5th-6th April until the end of the study on the 18th of May. We find that infections are concentrated in certain postcodes indicating that outbreaks of infection are extremely localised. In contrast, other areas remain comparatively untouched by the epidemic. ConclusionThese data indicate that sero-surveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic like the current SARS-CoV-2 outbreak.

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