HIV-1 prehairpin intermediate inhibitors show efficacy independent of neutralization tier.

HIV-1 strains are categorized into one of three neutralization tiers based on the relative ease by which they are neutralized by plasma from HIV-1-infected donors not on antiretroviral therapy; tier-1 strains are particularly sensitive to neutralization while tier-2 and tier-3 strains are increasingly difficult to neutralize. Most broadly neutralizing antibodies (bnAbs) previously described target the native prefusion conformation of HIV-1 Envelope (Env), but the relevance of the tiered categories for inhibitors targeting another Env conformation, the prehairpin intermediate, is not well understood. Here, we show that two inhibitors targeting distinct highly conserved regions of the prehairpin intermediate have strikingly consistent neutralization potencies (within ~100-fold for a given inhibitor) against strains in all three neutralization tiers of HIV-1; in contrast, best-in-class bnAbs targeting diverse Env epitopes vary by more than 10,000-fold in potency against these strains. Our results indicate that antisera-based HIV-1 neutralization tiers are not relevant for inhibitors targeting the prehairpin intermediate and highlight the potential for therapies and vaccine efforts targeting this conformation.

Converting non-neutralizing SARS-CoV-2 antibodies into broad-spectrum inhibitors.

Omicron and its subvariants have rendered most authorized monoclonal antibody-based treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ineffective, highlighting the need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective antibodies target variable epitopes. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to known non-neutralizing antibodies that target highly conserved epitopes in the viral spike protein. These inhibitors, called receptor-blocking conserved non-neutralizing antibodies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron. Neutralization potency is lost when the linker joining the binding and inhibitory ReconnAb components is severed. In addition, a bi-functional ReconnAb, made by linking ACE2 to a bi-specific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron and BA.2. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad-spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.

The high-affinity immunoglobulin receptor FcγRI potentiates HIV-1 neutralization via antibodies against the gp41 N-heptad repeat.

The HIV-1 gp41 N-heptad repeat (NHR) region of the prehairpin intermediate, which is transiently exposed during HIV-1 viral membrane fusion, is a validated clinical target in humans and is inhibited by the Food and Drug Administration (FDA)-approved drug enfuvirtide. However, vaccine candidates targeting the NHR have yielded only modest neutralization activities in animals; this inhibition has been largely restricted to tier-1 viruses, which are most sensitive to neutralization by sera from HIV-1-infected individuals. Here, we show that the neutralization activity of the well-characterized NHR-targeting antibody D5 is potentiated >5,000-fold in TZM-bl cells expressing FcγRI compared with those without, resulting in neutralization of many tier-2 viruses (which are less susceptible to neutralization by sera from HIV-1-infected individuals and are the target of current antibody-based vaccine efforts). Further, antisera from guinea pigs immunized with the NHR-based vaccine candidate (ccIZN36)3 neutralized tier-2 viruses from multiple clades in an FcγRI-dependent manner. As FcγRI is expressed on macrophages and dendritic cells, which are present at mucosal surfaces and are implicated in the early establishment of HIV-1 infection following sexual transmission, these results may be important in the development of a prophylactic HIV-1 vaccine.

A Derivative of the D5 Monoclonal Antibody That Targets the gp41 N-Heptad Repeat of HIV-1 with Broad Tier-2-Neutralizing Activity.

HIV-1 infection is initiated by the viral glycoprotein Env, which, after interaction with cellular coreceptors, adopts a transient conformation known as the prehairpin intermediate (PHI). The N-heptad repeat (NHR) is a highly conserved region of gp41 exposed in the PHI; it is the target of the FDA-approved drug enfuvirtide and of neutralizing monoclonal antibodies (mAbs). However, to date, these mAbs have only been weakly effective against tier-1 HIV-1 strains, which are most sensitive to neutralizing antibodies. Here, we engineered and tested 11 IgG variants of D5, an anti-NHR mAb, by recombining previously described mutations in four of D5's six antibody complementarity-determining regions. One variant, D5_AR, demonstrated 6-fold enhancement in the 50% inhibitory dose (ID50) against lentivirus pseudotyped with HXB2 Env. D5_AR exhibited weak cross-clade neutralizing activity against a diverse set of tier-2 HIV-1 viruses, which are less sensitive to neutralizing antibodies than tier-1 viruses and are the target of current antibody-based vaccine efforts. In addition, the neutralization potency of D5_AR IgG was greatly enhanced in target cells expressing FcγRI, with ID50 values of <0.1 µg/ml; this immunoglobulin receptor is expressed on macrophages and dendritic cells, which are implicated in the early stages of HIV-1 infection of mucosal surfaces. D5 and D5_AR have equivalent neutralization potency in IgG, Fab, and single-chain variable-fragment (scFv) formats, indicating that neutralization is not impacted by steric hindrance. Taken together, these results provide support for vaccine strategies that target the PHI by eliciting antibodies against the gp41 NHR and support investigation of anti-NHR mAbs in nonhuman primate passive immunization studies. IMPORTANCE Despite advances in antiretroviral therapy, HIV remains a global epidemic and has claimed more than 32 million lives. Accordingly, developing an effective HIV vaccine remains an urgent public health need. The gp41 N-heptad repeat (NHR) of the HIV-1 prehairpin intermediate (PHI) is highly conserved (>90%) and is inhibited by the FDA-approved drug enfuvirtide, making it an attractive vaccine target. However, to date, anti-NHR antibodies have not been potent. Here, we engineered D5_AR, a more potent variant of the anti-NHR antibody D5, and established its ability to inhibit HIV-1 strains that are more difficult to neutralize and are more representative of circulating strains (tier-2 strains). The neutralizing activity of D5_AR was greatly potentiated in cells expressing FcγRI; FcγRI is expressed on cells that are implicated at the earliest stages of sexual HIV-1 transmission. Taken together, these results bolster efforts to target the gp41 NHR and the PHI for vaccine development.

Neutralizing antibodies targeting the SARS-CoV-2 receptor binding domain isolated from a naïve human antibody library.

Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naïve antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naïve antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC50 values as low as 60 ng/ml in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naïve antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.

Crystal structure of AdoMet radical enzyme 7-carboxy-7-deazaguanine synthase from Escherichia coli suggests how modifications near [4Fe-4S] cluster engender flavodoxin specificity.

7-Carboxy-7-deazaguanine synthase, QueE, catalyzes the radical mediated ring contraction of 6-carboxy-5,6,7,8-tetrahydropterin, forming the characteristic pyrrolopyrimidine core of all 7-deazaguanine natural products. QueE is a member of the S-adenosyl-L-methionine (AdoMet) radical enzyme superfamily, which harnesses the reactivity of radical intermediates to perform challenging chemical reactions. Members of the AdoMet radical enzyme superfamily utilize a canonical binding motif, a CX3 CXϕC motif, to bind a [4Fe-4S] cluster, and a partial (ß/α)6 TIM barrel fold for the arrangement of AdoMet and substrates for catalysis. Although variations to both the cluster-binding motif and the core fold have been observed, visualization of drastic variations in the structure of QueE from Burkholderia multivorans called into question whether a re-haul of the defining characteristics of this superfamily was in order. Surprisingly, the structure of QueE from Bacillus subtilis revealed an architecture more reminiscent of the classical AdoMet radical enzyme. With these two QueE structures revealing varying degrees of alterations to the classical AdoMet fold, a new question arises: what is the purpose of these alterations? Here, we present the structure of a third QueE enzyme from Escherichia coli, which establishes the middle range of the spectrum of variation observed in these homologs. With these three homologs, we compare and contrast the structural architecture and make hypotheses about the role of these structural variations in binding and recognizing the biological reductant, flavodoxin. Broader impact statement: We know more about how enzymes are tailored for catalytic activity than about how enzymes are tailored to react with a physiological reductant. Here, we consider structural differences between three 7-carboxy-7-deazaguanine synthases and how these differences may be related to the interaction between these enzymes and their biological reductant, flavodoxin.