Abstract A potential single-shot HIV therapy may be transplanted engineered B cells allowing strong secretion of broadly neutralizing antibodies (bNAbs). However, extensive, and expensive ex-vivo manipulations performed in specialized facilities hinders clinical potential of this approach. Furthermore, allogeneic B cell therapy necessitates MHC-II compatibility to receive mandatory T-cell help. To overcome these limitations, we engineer B cells in-vivo. In particular, we demonstrate that a single, systemic dose of dual AAV, one coding for CRISPR/Cas9 and another coding for a bNAb donor cassette, allows for site specific integration in B cells. Following immunizations, we show memory retention and bNAb secretion at high titers. Antibodies secreted by the engineered B cells were found to be of multiple isotypes and IgGs could neutralize autologous and heterologous pseudoviruses. We found engineered B cell subsets in the spleen and blood. We detected homing of in-vivo engineered cells to germinal centers and bone marrow. Biodistribution of the donor AAV over time and as compared to a CRISPR- group, indicated expansion of engineered B cells in lymphatic tissues. We determined minimal CRISPR/Cas9 off-target cleavage, using unbiased, highly sensitive, CHANGE-Seq analysis. Finally, we diminished on-target, non-productive double-strand breaks at undesired tissues by expressing Cas9 from a B cell specific promoter. Eliciting a specific, neutralizing serological response to hypervariable viruses is a long-standing challenge in medicine. B cell engineering provides an opportunity to express therapeutic antibodies to generate an adaptive and evolving immunity.
ABSTRACT The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.
ABSTRACT Extensive studies with subtype A BG505-derived HIV envelope glycoprotein (Env) SOSIP immunogens have revealed that the dominant autologous neutralizing site in rabbits is located in an exposed region of the heavily glycosylated trimer that lacks potential N-linked glycosylation sites at positions 230, 241, and 289. The Env derived from B41, a subtype B virus, shares a glycan hole centered on positions 230 and 289. BG505 and B41 SOSIP immunogens were combined to test whether immunization in rabbits could induce broader Tier 2 neutralizing responses to the common glycan hole shared between BG505 and B41. Here we isolated autologous neutralizing antibodies (nAbs) that were induced by immunization with B41 SOSIP alone, as well as B41 and BG505 co-immunization, and describe their structure in complex with the B41 SOSIP trimer. Our data suggest that distinct autologous nAb lineages are induced by BG505 and B41 immunogens, even when both immunogens were administered together. In contrast to previously described BG505 glycan hole antibodies, the B41-specific nAbs accommodate the highly conserved N241 glycan (>97% conserved), which is present in B41. Single particle cryo-electron microscopy (cryoEM) studies confirmed that B41 and BG505-specific nAbs bind to overlapping glycan hole epitopes. In an attempt to broaden the reactivity of a B41-specific nAb, mutations in the BG505 glycan hole epitope guided by our high-resolution data only recovered partial binding. Overall, designing prime-boost immunogens to increase the breath of nAb responses directed at glycan holes epitopes remains challenging even when the typically immunodominant glycan holes despite overlap with different Envs. IMPORTANCE A glycan hole is one of the most dominant autologous neutralizing epitopes targeted on BG505 and B41 SOSIP trimer immunized rabbits. Our high-resolution cryoEM studies of B41 in complex with a B41-specific antibody complex elucidate the molecular basis of this strain-specific glycan hole response. We conclude that eliciting cross-reactive responses to this region would likely require hybrid immunogens that bridge between BG505 and B41.
We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody, PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved anti-HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses.
The recurrent zoonotic spillover of coronaviruses (CoVs) into the human population underscores the need for broadly active countermeasures. Here, we employed a directed evolution approach to engineer three SARS-CoV-2 antibodies for enhanced neutralization breadth and potency. One of the affinity-matured variants, ADG-2, displays strong binding activity to a large panel of sarbecovirus receptor binding domains (RBDs) and neutralizes representative epidemic sarbecoviruses with remarkable potency. Structural and biochemical studies demonstrate that ADG-2 employs a unique angle of approach to recognize a highly conserved epitope overlapping the receptor binding site. In murine models of SARS-CoV and SARS-CoV-2 infection, passive transfer of ADG-2 provided complete protection against respiratory burden, viral replication in the lungs, and lung pathology. Altogether, ADG-2 represents a promising broad-spectrum therapeutic candidate for the treatment and prevention of SARS-CoV-2 and future emerging SARS-like CoVs.
Abstract Pre-existing immunity to seasonal endemic coronaviruses could have profound consequences for antibody responses to SARS-CoV-2, induced from natural infection or vaccination. A first step to establish whether pre-existing responses can impact SARS-CoV-2 infection is to understand the nature and extent of cross-reactivity in humans to coronaviruses. Here we compare serum antibody and memory B cell responses to coronavirus spike proteins from pre-pandemic and SARS-CoV-2 convalescent donors using binding and functional assays. We show weak evidence of pre-existing SARS-CoV-2 cross-reactive serum antibodies in pre-pandemic donors. However, we find evidence of pre-existing cross-reactive memory B cells that are activated during SARS-CoV-2 infection. Monoclonal antibodies show varying degrees of cross-reactivity with betacoronaviruses, including SARS-CoV-1 and endemic coronaviruses. We identify one cross-reactive neutralizing antibody specific to the S2 subunit of the S protein. Our results suggest that pre-existing immunity to endemic coronaviruses should be considered in evaluating antibody responses to SARS-CoV-2.
