The SARS-CoV-2 neutralizing antibody response to SD1 and its evasion by BA.2.86

Under pressure from neutralising antibodies induced by vaccination or infection the SARS-CoV-2 spike gene has become a hotspot for evolutionary change, leading to the failure of all mAbs developed for clinical use. Most potent antibodies bind to the receptor binding domain which has become heavily mutated. Here we study responses to a conserved epitope in sub-domain-1 (SD1) of spike which have become more prominent because of mutational escape from antibodies directed to the receptor binding domain. Some SD1 reactive mAbs show potent and broad neutralization of SARS-CoV-2 variants. We structurally map the dominant SD1 epitope and provide a mechanism of action by blocking interaction with ACE2. Mutations in SD1 have not been sustained to date, but one, E554K, leads to escape from mAbs. This mutation has now emerged in several sublineages including BA.2.86, reflecting selection pressure on the virus exerted by the increasing prominence of the anti-SD1 response

Emerging variants develop total escape from potent monoclonal antibodies induced by BA.4/5 infection

The rapid evolution of SARS-CoV-2 is driven in part by a need to evade the antibody response in the face of high levels of immunity. Here, we isolate spike (S) binding monoclonal antibodies (mAbs) from vaccinees who suffered vaccine break-through infections with Omicron sub lineages BA.4 or BA.5. Twenty eight potent antibodies are isolated and characterised functionally, and in some cases structurally. Since the emergence of BA.4/5, SARS-CoV-2 has continued to accrue mutations in the S protein, to understand this we characterize neutralization of a large panel of variants and demonstrate a steady attrition of neutralization by the panel of BA.4/5 mAbs culminating in total loss of function with recent XBB.1.5.70 variants containing the so-called ‘FLip’ mutations at positions 455 and 456. Interestingly, activity of some mAbs is regained on the recently reported variant BA.2.86

A structure-function analysis shows SARS-CoV-2 BA.2.86 balances antibody escape and ACE2 affinity.

BA.2.86, a recently described sublineage of SARS-CoV-2 Omicron, contains many mutations in the spike gene. It appears to have originated from BA.2 and is distinct from the XBB variants responsible for many infections in 2023. The global spread and plethora of mutations in BA.2.86 has caused concern that it may possess greater immune-evasive potential, leading to a new wave of infection. Here, we examine the ability of BA.2.86 to evade the antibody response to infection using a panel of vaccinated or naturally infected sera and find that it shows marginally less immune evasion than XBB.1.5. We locate BA.2.86 in the antigenic landscape of recent variants and look at its ability to escape panels of potent monoclonal antibodies generated against contemporary SARS-CoV-2 infections. We demonstrate, and provide a structural explanation for, increased affinity of BA.2.86 to ACE2, which may increase transmissibility

A delicate balance between antibody evasion and ACE2 affinity for Omicron BA.2.75

Variants of SARS CoV-2 have caused successive global waves of infection. These variants, with multiple mutations in the spike protein are thought to facilitate escape from natural and vaccine-induced immunity and often increase in the affinity for ACE2. The latest variant to cause concern is BA.2.75, identified in India where it is now the dominant strain, with evidence of wider dissemination. BA.2.75 is derived from BA.2 and contains four additional mutations in the receptor binding domain (RBD). Here we perform an antigenic and biophysical characterization of BA.2.75, revealing an interesting balance between humoral evasion and ACE2 receptor affinity. ACE2 affinity for BA.2.75 is increased 9-fold compared to BA.2; there is also evidence of escape of BA.2.75 from immune serum, particularly that induced by Delta infection which may explain the rapid spread in India, where BA.2.75 is now the dominant variant. ACE2 affinity appears to be prioritised over greater escape

Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum

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

Synergistic effect of high-mobility group box-1 and lipopolysaccharide on cytokine induction in bovine peripheral blood mononuclear cells

High-mobility group box 1 (HMGB1) is one of the potent endogenous adjuvants released by necrotic and activated innate immune cells. HMGB1 modulates innate and adaptive immune responses in humans and mice by mediating immune cells crosstalk. However, the immuno-modulatory effects of HMGB1 in the bovine immune system are not clearly known. In this study, the effect of bovine HMGB1 alone or in combination with LPS on the expression kinetics of cytokines upon in vitro stimulation of bovine peripheral blood mononuclear cells (PBMCs) was investigated by quantitative PCR assay. The biological activity of bovine HMGB1 expressed in this prokaryotic expression system was confirmed by its ability to induce nitric oxide secretion in RAW 264.7 cells. The present results indicate that HMGB1 induces a more delayed TNF-α response than does LPS in stimulated PBMCs. However, IFN-γ, IFN-β and IL-12 mRNA transcription peaked at 6 hr post stimulation after both treatments. Further, HMGB1 and LPS heterocomplex up-regulated TNF-α, IFN-γ and IL-12 mRNA expression significantly than did individual TLR4 agonists. The heterocomplex also enhanced the expression of TLR4 on bovine PBMCs. In conclusion, the data indicate that HMGB1 and LPS act synergistically and enhance proinflammatory cytokines, thereby eliciting Th1 responses in bovine PBMCs. These results suggest that HMGB1 can act as an adjuvant in modulating the bovine immune system and thus lays a foundation for using HMGB1 as an adjuvant in various bovine vaccine preparations

Rapid Detection of Peste des Petits Ruminants Virus (PPRV) Nucleic Acid Using a Novel Low-Cost Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Future Use in Nascent PPR Eradication Programme

Peste des petits ruminants (PPR) is a disease of small ruminants caused by peste des petits ruminants virus (PPRV), and is endemic in Asia, the Middle East and Africa. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) have improved the sensitivity and rapidity of diagnosing PPR. However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for PPR would improve the fast implementation of control policies, particularly when PPR has been targeted to be eradicated by 2030. Loop-mediated isothermal amplification (LAMP) assays are simple to use, rapid, and have sensitivity and specificity within the range of RT-qPCR; and can be performed in the field using disposable consumables and portable equipment. This study describes the development of a novel RT-LAMP assay for the detection of PPRV nucleic acid by targeting the N-protein gene. The RT-LAMP assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. The test displayed 100% concordance with RT-qPCR when considering an RT-qPCR cut-off value of CT >40. Further, the RT-LAMP assay was evaluated using experimental and outbreak samples without prior RNA extraction making it more time and cost-effective. This assay provides a solution for a pen-side, rapid and inexpensive PPR diagnostic for use in the field in nascent PPR eradication programme

Effective Interferon Lambda Treatment Regimen To Control Lethal MERS-CoV Infection in Mice.

Effective broad-spectrum antivirals are critical to prevent and control emerging human coronavirus (hCoV) infections. Despite considerable progress made toward identifying and evaluating several synthetic broad-spectrum antivirals against hCoV infections, a narrow therapeutic window has limited their success. Enhancing the endogenous interferon (IFN) and IFN-stimulated gene (ISG) response is another antiviral strategy that has been known for decades. However, the side effects of pegylated type-I IFNs (IFN-Is) and the proinflammatory response detected after delayed IFN-I therapy have discouraged their clinical use. In contrast to IFN-Is, IFN-λ, a dominant IFN at the epithelial surface, has been shown to be less proinflammatory. Consequently, we evaluated the prophylactic and therapeutic efficacy of IFN-λ in hCoV-infected airway epithelial cells and mice. Human primary airway epithelial cells treated with a single dose of IFN-I (IFN-α) and IFN-λ showed similar ISG expression, whereas cells treated with two doses of IFN-λ expressed elevated levels of ISG compared to that of IFN-α-treated cells. Similarly, mice treated with two doses of IFN-λ were better protected than mice that received a single dose, and a combination of prophylactic and delayed therapeutic regimens completely protected mice from a lethal Middle East respiratory syndrome CoV (MERS-CoV) infection. A two-dose IFN-λ regimen significantly reduced lung viral titers and inflammatory cytokine levels with marked improvement in lung inflammation. Collectively, we identified an effective regimen for IFN-λ use and demonstrated the protective efficacy of IFN-λ in MERS-CoV-infected mice. IMPORTANCE Effective antiviral agents are urgently required to prevent and treat individuals infected with SARS-CoV-2 and other emerging viral infections. The COVID-19 pandemic has catapulted our efforts to identify, develop, and evaluate several antiviral agents. However, a narrow therapeutic window has limited the protective efficacy of several broad-spectrum and CoV-specific antivirals. IFN-λ is an antiviral agent of interest due to its ability to induce a robust endogenous antiviral state and low levels of inflammation. Here, we evaluated the protective efficacy and effective treatment regimen of IFN-λ in mice infected with a lethal dose of MERS-CoV. We show that while prophylactic and early therapeutic IFN-λ administration is protective, delayed treatment is detrimental. Notably, a combination of prophylactic and delayed therapeutic administration of IFN-λ protected mice from severe MERS. Our results highlight the prophylactic and therapeutic use of IFN-λ against lethal hCoV and likely other viral lung infections

Isolation of a pair of potent broadly neutralizing mAb binding to RBD and SD1 domains of SARS-CoV-2

Commercially developed monoclonal antibodies (mAb) have been effective in the prevention or treatment of SARS-CoV-2 infection1-3 but the rapid antigenic evolution of the Omicron sub-lineages has reduced their activity4-8 and they are no longer licensed for use in many countries. Here, we isolate spike binding monoclonal antibodies from vaccinees who suffered vaccine break-through infections with Omicron sublineages BA.4/5. We find that it is possible for antibodies targeting highly mutated regions to recover broad activity through allosteric effects (mAb BA.4/5-35) and characterise a pair of potent mAbs with extremely broad neutralization against current and historical SARS-CoV-2 variants. One, mAb BA.4/5-2, binds at the back of the left shoulder of the receptor binding domain (RBD) in an area which has resisted mutational change to date. The second, mAb BA.4/5-5, binds a conserved epitope in sub-domain 1 (SD1). The isolation of this pair of antibodies with non-overlapping epitopes shows that potent and extremely broadly neutralizing antibodies are still generated following infection and SD1 directed mAbs may increase the resilience of mAb therapeutics/prophylactics against SARS-CoV-2

Rapid escape of new SARS-CoV-2 Omicron variants from BA.2 directed antibody responses

In November 2021 Omicron BA.1, containing a raft of new spike mutations emerged and quickly spread globally. Intense selection pressure to escape the antibody response produced by vaccines or SARS-CoV-2 infection then led to a rapid succession of Omicron sub-lineages with waves of BA.2 then BA.4/5 infection. Recently, many variants have emerged such as BQ.1 and XBB, which carry up to 8 additional RBD amino-acid substitutions compared to BA.2. We describe a panel of 25 potent mAbs generated from vaccinees suffering BA.2 breakthrough infections. Epitope mapping shows potent mAb binding shifting to 3 clusters, 2 corresponding to early-pandemic binding hotspots. The RBD mutations in recent variants map close to these binding sites and knock out or severely knock down neutralization activity of all but 1 potent mAb. This recent mAb escape corresponds with large falls in neutralization titre of vaccine or BA.1, BA.2 or BA.4/5 immune serum