Generation of SARS-CoV-2 escape mutations by monoclonal antibody therapy.

COVID-19 patients at risk of severe disease may be treated with neutralising monoclonal antibodies (mAbs). To minimise virus escape from neutralisation these are administered as combinations e.g. casirivimab+imdevimab or, for antibodies targeting relatively conserved regions, individually e.g. sotrovimab. Unprecedented genomic surveillance of SARS-CoV-2 in the UK has enabled a genome-first approach to detect emerging drug resistance in Delta and Omicron cases treated with casirivimab+imdevimab and sotrovimab respectively. Mutations occur within the antibody epitopes and for casirivimab+imdevimab multiple mutations are present on contiguous raw reads, simultaneously affecting both components. Using surface plasmon resonance and pseudoviral neutralisation assays we demonstrate these mutations reduce or completely abrogate antibody affinity and neutralising activity, suggesting they are driven by immune evasion. In addition, we show that some mutations also reduce the neutralising activity of vaccine-induced serum.

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. Graphical Dijokaite-Guraliuc et al. analyse potently neutralizing antibodies from vaccinated individuals with BA.2 breakthrough infections. The antibodies bind 3 sites on the receptor binding domain, 2 in common with early pandemic antibodies. Mutations in more recent variants map closely to these sites leading to reduced neutralization in all but one mAb.

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 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection then led to a rapid succession of Omicron sub-lineages with waves of BA.2 and then BA.4/5 infection. Recently, many variants have emerged such as BQ.1 and XBB, which carry up to 8 additional receptor-binding domain (RBD) amino acid substitutions compared with BA.2. We describe a panel of 25 potent monoclonal antibodies (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 titer of vaccine or BA.1, BA.2, or BA.4/5 immune serum.

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

Variants of severe acute respiratory syndrome coronavirus 2 (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 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 with 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 where there is a high background of Delta infection. ACE2 affinity appears to be prioritized over greater escape.

Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infections During SARS-CoV-2.

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate infection with the SARS-CoV-2 USA-WA1/2020 strain increased the risk of pneumococcal (type 2 strain D39) coinfection in a time-dependent, but sex-independent, manner in the transgenic K18-hACE2 mouse model of COVID-19. Bacterial coinfection increased lethality when the bacteria was initiated at 5 or 7 d post-virus infection (pvi) but not at 3 d pvi. Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.

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, which was first described in November 2021, spread rapidly to become globally dominant and has split into a number of sublineages. 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 sublineages, 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, they contain further mutations in the receptor-binding domain of their spikes. 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 the serum from individuals vaccinated with triple doses of AstraZeneca or Pfizer vaccine compared with BA.1 and BA.2. Furthermore, using the 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.

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.

Alveolar macrophages protect mice from MERS-CoV-induced pneumonia and severe disease.

Emerging and re-emerging human coronaviruses (hCoVs) cause severe respiratory illness in humans, but the basis for lethal pneumonia in these diseases is not well understood. Alveolar macrophages (AMs) are key orchestrators of host antiviral defense and tissue tolerance during a variety of respiratory infections, and AM dysfunction is associated with severe COVID-19. In this study, using a mouse model of Middle East respiratory syndrome coronavirus (MERS-CoV) infection, we examined the role of AMs in MERS pathogenesis. Our results show that depletion of AMs using clodronate (CL) liposomes significantly increased morbidity and mortality in human dipeptidyl peptidase 4 knock-in (hDPP4-KI) mice. Detailed examination of control and AM-depleted lungs at different days postinfection revealed increased neutrophil activity but a significantly reduced MERS-CoV-specific CD4 T-cell response in AM-deficient lungs during later stages of infection. Furthermore, enhanced MERS severity in AM-depleted mice correlated with lung inflammation and lesions. Collectively, these data demonstrate that AMs are critical for the development of an optimal virus-specific T-cell response and controlling excessive inflammation during MERS-CoV infection.