SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive ...possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the 'up' and 'down' conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.
After a prolonged period of near-complete global dominance of the recombinant XBB family of SARS-CoV-2 lineages, a substantially mutated sublineage of BA.2 has recently emerged. With a long branch of ...unobserved evolution (figure A), including more than 30 mutations in the spike protein relative to BA.2, with many at key antigenic sites (figure B), the emergence of BA.2.86 is reminiscent of the initial emergence of omicron.2 This raises immediate questions about whether it is sensitive to any previously approved clinical monoclonal antibodies with activity against BA.2, and the degree to which it is able to escape antibody responses in the current setting in which individual exposure histories are a complex combination of multiple immunisations and multiple previous SARS-CoV-2 infections. To characterise the relative sensitivity of BA.2.86 to current antibody-mediated immunity at the population level, we evaluated BA.2.86 neutralisation by serum sampled from random blood donors in Stockholm, Sweden, during week 34 (Aug 21–27), 2023.
The outbreak of the SARS-CoV-2 virus and its rapid spread into a global pandemic made the urgent development of scalable vaccines to prevent coronavirus disease (COVID-19) a global health and ...economic imperative. Here, we characterized and compared the immunogenicity of two alphavirus-based DNA-launched self-replicating (DREP) vaccine candidates encoding either SARS-CoV-2 spike glycoprotein (DREP-S) or a spike ectodomain trimer stabilized in prefusion conformation (DREP-S
). We observed that the two DREP constructs were immunogenic in mice inducing both binding and neutralizing antibodies as well as T cell responses. Interestingly, the DREP coding for the unmodified spike turned out to be more potent vaccine candidate, eliciting high titers of SARS-CoV-2 specific IgG antibodies that were able to efficiently neutralize pseudotyped virus after a single immunization. In addition, both DREP constructs were able to efficiently prime responses that could be boosted with a heterologous spike protein immunization. These data provide important novel insights into SARS-CoV-2 vaccine design using a rapid response DNA vaccine platform. Moreover, they encourage the use of mixed vaccine modalities as a strategy to combat SARS-CoV-2.
Here we report the sensitivity of emerging omicron sublineages BA.2.75.2, BA.4.6, and BA.2.10.4 to neutralisation by a panel of clinically relevant and preclinical monoclonal antibodies and by serum ...from blood donated in Stockholm, Sweden. To characterise the evolving resistance to immunity at the population level, we evaluated the sensitivity to neutralisation by serum from random blood donors in Stockholm, including a cohort sampled Nov 8–14, 2021 (November 2021 cohort; figure C), before the emergence of the omicron variant; a cohort sampled April 11–17, 2022 (April 2022 cohort; figure D), after both a large wave of infections (driven by BA.1 then BA.2) and the rollout of third vaccine doses; and a cohort sampled Aug 29 to Sept 4, 2022 (September 2022 cohort; figure E), after the spread of BA.5. Taken together, these data identify profound antibody escape by the emerging omicron sublineage BA.2.75.2, suggesting that it effectively evades current humoral immunity in the population.
The SARS-CoV-2 omicron (B.1.1.529) variant, which was first identified in November, 2021, spread rapidly in many countries, with a spike protein highly diverged from previously known variants, and ...raised concerns that this variant might evade neutralising antibody responses. We therefore aimed to characterise the sensitivity of the omicron variant to neutralisation.
For this cross-sectional study, we cloned the sequence encoding the omicron spike protein from a diagnostic sample to establish an omicron pseudotyped virus neutralisation assay. We quantified the neutralising antibody ID50 (the reciprocal dilution that produces 50% inhibition) against the omicron spike protein, and the fold-change in ID50 relative to the spike of wild-type SARS-CoV-2 (ie, the pandemic founder variant), for one convalescent reference plasma pool (WHO International Standard for anti-SARS-CoV-2 immunoglobulin 20/136), three reference serum pools from vaccinated individuals, and two cohorts from Stockholm, Sweden: one comprising previously infected hospital workers (17 sampled in November, 2021, after vaccine rollout and nine in June or July, 2020, before vaccination) and one comprising serum from 40 randomly sampled blood donors donated during week 48 (Nov 29–Dec 5) of 2021. Furthermore, we assessed the neutralisation of omicron by five clinically relevant monoclonal antibodies (mAbs).
Neutralising antibody responses in reference sample pools sampled shortly after infection or vaccination were substantially less potent against the omicron variant than against wild-type SARS-CoV-2 (seven-fold to 42-fold reduction in ID50 titres). Similarly, for sera obtained before vaccination in 2020 from a cohort of convalescent hospital workers, neutralisation of the omicron variant was low to undetectable (all ID50 titres <20). However, in serum samples obtained in 2021 from two cohorts in Stockholm, substantial cross-neutralisation of the omicron variant was observed. Sera from 17 hospital workers after infection and subsequent vaccination had a reduction in average potency of only five-fold relative to wild-type SARS-CoV-2 (geometric mean ID50 titre 495 vs 105), and two donors had no reduction in potency. A similar pattern was observed in randomly sampled blood donors (n=40), who had an eight-fold reduction in average potency against the omicron variant compared with wild-type SARS-CoV-2 (geometric mean ID50 titre 369 vs 45). We found that the omicron variant was resistant to neutralisation (50% inhibitory concentration IC50 >10 μg/mL) by mAbs casirivimab (REGN-10933), imdevimab (REGN-10987), etesevimab (Ly-CoV016), and bamlanivimab (Ly-CoV555), which form part of antibody combinations used in the clinic to treat COVID-19. However, S309, the parent of sotrovimab, retained most of its activity, with only an approximately two-fold reduction in potency against the omicron variant compared with ancestral D614G SARS-CoV-2 (IC50 0·1–0·2 μg/mL).
These data highlight the extensive, but incomplete, evasion of neutralising antibody responses by the omicron variant, and suggest that boosting with licensed vaccines might be sufficient to raise neutralising antibody titres to protective levels.
European Union Horizon 2020 research and innovation programme, European and Developing Countries Clinical Trials Partnership, SciLifeLab, and the Erling-Persson Foundation.
G446S has been predicted to be a site of potential escape from antibodies elicited by current vaccines that still neutralise omicron.5 Furthermore, it has been identified as a site of potential ...escape from LY-CoV1404 (bebtelovimab), which represents one of the last remaining classes of first-generation monoclonal antibodies that can cross-neutralise BA.2, BA.4, and BA.5.1 As waves of omicron infections have occurred in many countries, identifying the sensitivity of newly emerging variants to neutralisation by sera sampled subsequent to these waves is required to inform public health policy. Here we report the sensitivity of the BA.2.75 spike protein to neutralisation by a panel of clinically relevant and preclinical monoclonal antibodies, as well as by serum from blood donated in Stockholm, Sweden, during Nov 8–14, 2021 (n=20), and April 11–17, 2022 (n=20). The relative sensitivity of BA.2.75 in these cohorts of blood donors is largely concordant with those seen in vaccinated individuals (CoronaVac; Sinovac Life Sciences, Beijing, China) with and without BA.1 or BA.2 breakthrough infection.7 As infection histories become more complex, and a large proportion of infections go undetected, monitoring of population-level immunity from random samples is increasingly crucial for understanding and contextualising the immune evasion properties of new SARS-CoV-2 variants.
The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional ...antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC
of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.
Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been ...unsuccessful, a minority of individuals naturally develop these antibodies after many years of infection. How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1-infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strain-specific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly under-represented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved BCN antibody epitopes.
Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased ...valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC
in the low picomolar range.
Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental ...pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.
PDF
