Covalent penicillin-protein conjugates elicit anti-drug antibodies that are clonally and functionally restricted.

Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although ß-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical ß-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)-regardless of CDRH3 length-limits cross-reactivity against other ß-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the 'germline-guided reverse engineering' of such drugs away from unwanted immune responses.

Apoptosis-mediated ADAM10 activation removes a mucin barrier promoting T cell efferocytosis.

Efferocytic clearance of apoptotic cells in general, and T cells in particular, is required for tissue and immune homeostasis. Transmembrane mucins are extended glycoproteins highly expressed in the cell glycocalyx that function as a barrier to phagocytosis. Whether and how mucins may be regulated during cell death to facilitate efferocytic corpse clearance is not well understood. Here we show that normal and transformed human T cells express a subset of mucins which are rapidly and selectively removed from the cell surface during apoptosis. This process is mediated by the ADAM10 sheddase, the activity of which is associated with XKR8-catalyzed flipping of phosphatidylserine to the outer leaflet of the plasma membrane. Mucin clearance enhances uptake of apoptotic T cells by macrophages, confirming mucins as an enzymatically-modulatable barrier to efferocytosis. Together these findings demonstrate a glycocalyx regulatory pathway with implications for therapeutic intervention in the clearance of normal and transformed apoptotic T cells.

Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens.

Chemical cross-linking is used to stabilize protein structures with additional benefits of pathogen and toxin inactivation for vaccine use, but its use has been restricted by the potential for local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for inducing a biological outcome such as the elicitation of antibodies to conformationally sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) to cross-link two soluble Env trimers, selected well-folded trimer species using antibody affinity, and transferred this process to good manufacturing practice (GMP) for experimental medicine use. Cross-linking enhanced trimer stability to biophysical and enzyme attack. Cryo-EM analysis revealed that cross-linking retained the overall structure with root-mean-square deviations (RMSDs) between unmodified and cross-linked Env trimers of 0.4-0.5 Å. Despite this negligible distortion of global trimer structure, we identified individual inter-subunit, intra-subunit, and intra-protomer cross-links. Antigenicity and immunogenicity of the trimers were selectively modified by cross-linking, with cross-linked ConS retaining bnAb binding more consistently than ConM. Thus, the EDC cross-linking process improves trimer stability whilst maintaining protein folding, and is readily transferred to GMP, consistent with the more general use of this approach in protein-based vaccine design.

Intranasal SARS-CoV-2 spike-based immunisation adjuvanted with polyethyleneimine elicits mucosal and systemic humoral responses in mice.

The SARS-CoV-2 pandemic continues despite the presence of effective vaccines, and novel vaccine approaches may help to reduce viral spread and associated COVID-19 disease. Current vaccine administration modalities are based on systemic needle-administered immunisation which may be suboptimal for mucosal pathogens. Here we demonstrate in a mouse model that small-volume intranasal administration of purified spike (S) protein in the adjuvant polyethylenemine (PEI) elicits robust antibody responses with modest systemic neutralisation activity. Further, we test a heterologous intranasal immunisation regimen, priming with S and boosting with RBD-Fc. Our data identify small volume PEI adjuvantation as a novel platform with potential for protective mucosal vaccine development.

Pathogen-sugar interactions revealed by universal saturation transfer analysis.

Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily modified pathogen proteins can be confounded by overlapping sugar signals and/or compounded with known experimental constraints. Universal saturation transfer analysis (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin-lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an "end-on" manner. uSTA-guided modeling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar binding in SARS-CoV-2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins deep in the human lung as potentially relevant to virulence and/or zoonosis.

Shared sugars - parasite glycan homology in HIV-1 vaccine design.

Immune tolerance to self-glycans is a host mechanism to avoid autoimmunity, which is exploited by HIV-1 coating its envelope glycoproteins with glycans to evade neutralising antibodies (nAbs). Huettner et al. describe cross-reactivity between Schistosoma mansoni glycans and HIV-1 envelope glycoprotein glycans, suggesting a strategy for induction of HIV-1 nAbs by vaccination.

High thermostability improves neutralizing antibody responses induced by native-like HIV-1 envelope trimers.

Soluble HIV-1 envelope glycoprotein (Env) immunogens are a prime constituent of candidate vaccines designed to induce broadly neutralizing antibodies. Several lines of evidence suggest that enhancing Env immunogen thermostability can improve neutralizing antibody (NAb) responses. Here, we generated BG505 SOSIP.v9 trimers, which displayed virtually no reactivity with non-neutralizing antibodies and showed increased global and epitope thermostability, compared to previous BG505 SOSIP versions. Chemical crosslinking of BG505 SOSIP.v9 further increased the melting temperature to 91.3 °C, which is almost 25 °C higher than that of the prototype SOSIP.664 trimer. Next, we compared the immunogenicity of a palette of BG505-based SOSIP trimers with a gradient of thermostabilities in rabbits. We also included SOSIP.v9 proteins in which a strain-specific immunodominant epitope was masked by glycans to redirect the NAb response to other subdominant epitopes. We found that increased trimer thermostability correlated with increased potency and consistency of the autologous NAb response. Furthermore, glycan masking steered the NAb response to subdominant epitopes without decreasing the potency of the autologous NAb response. In summary, SOSIP.v9 trimers and their glycan masked versions represent an improved platform for HIV-1 Env based vaccination strategies.

Glycans in HIV-1 vaccine design - engaging the shield.

Glycans are repeating carbohydrate structures added as post-translational modifications (PTMs) to proteins, forming glycoproteins. Self-glycans found on human cells, and viral glycoproteins produced in host cells, are generally weakly immunogenic, which is necessary to avoid autoimmunity. This feature is exploited by many pathogenic viruses, which glycosylate surface proteins to evade or reduce immune recognition. The HIV type-1 (HIV-1) envelope glycoprotein (Env) is heavily glycosylated, which broadly acts to shield neutralisation-relevant protein surfaces with immunorecessive self-glycans to hinder B cell recognition. However, a small subset of HIV-1-infected individuals develops potent broadly neutralising antibodies (bnAbs), many of which directly engage the glycan shield. This provides hope that such antibodies could be elicited via vaccination and help to provide protective immunity. However, HIV-1 vaccine candidates have thus far failed to fully recapitulate such glycan-specific neutralising responses. In this review we consider the fundamental glycoimmunology and structural biology that underpin glycans in antibody evasion and as antibody targets and discuss potential approaches to harness glycan targeting for HIV-1 vaccine design.

Human MAIT cells respond to and suppress HIV-1.

Human MAIT cells sit at the interface between innate and adaptive immunity, are polyfunctional and are capable of killing pathogen infected cells via recognition of the Class IB molecule MR1. MAIT cells have recently been shown to possess an antiviral protective role in vivo and we therefore sought to explore this in relation to HIV-1 infection. There was marked activation of MAIT cells in vivo in HIV-1-infected individuals, which decreased following ART. Stimulation of THP1 monocytes with R5 tropic HIVBAL potently activated MAIT cells in vitro. This activation was dependent on IL-12 and IL-18 but was independent of the TCR. Upon activation, MAIT cells were able to upregulate granzyme B, IFNγ and HIV-1 restriction factors CCL3, 4, and 5. Restriction factors produced by MAIT cells inhibited HIV-1 infection of primary PBMCs and immortalized target cells in vitro. These data reveal MAIT cells to be an additional T cell population responding to HIV-1, with a potentially important role in controlling viral replication at mucosal sites.

Immunological and pathological outcomes of SARS-CoV-2 challenge following formalin-inactivated vaccine in ferrets and rhesus macaques.

There is an urgent requirement for safe and effective vaccines to prevent COVID-19. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferrets and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen 7 days after infection in ferrets. This increased lung pathology was observed at day 7 but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity.

Interplay of diverse adjuvants and nanoparticle presentation of native-like HIV-1 envelope trimers.

The immunogenicity of HIV-1 envelope (Env) trimers is generally poor. We used the clinically relevant ConM SOSIP trimer to compare the ability of different adjuvants (squalene emulsion, ISCOMATRIX, GLA-LSQ, and MPLA liposomes) to support neutralizing antibody (NAb) responses in rabbits. The trimers were administered as free proteins or on nanoparticles. The rank order for the adjuvants was ISCOMATRIX > SE > GLA-LSQ ~ MPLA liposomes > no adjuvant. Stronger NAb responses were elicited when the ConM SOSIP trimers were presented on ferritin nanoparticles. We also found that the GLA-LSQ adjuvant induced an unexpectedly strong antibody response to the ferritin core of the nanoparticles. This "off-target" effect may have compromised its ability to induce the more desired antitrimer antibodies. In summary, both adjuvants and nanoparticle display can improve the magnitude of the antibody response to SOSIP trimers but the best combination of trimer presentation and adjuvant can only be identified experimentally.

Disassembly of HIV envelope glycoprotein trimer immunogens is driven by antibodies elicited via immunization.

Rationally designed protein subunit vaccines are being developed for a variety of viruses including influenza, RSV, SARS-CoV-2, and HIV. These vaccines are based on stabilized versions of the primary targets of neutralizing antibodies on the viral surface, namely, viral fusion glycoproteins. While these immunogens display the epitopes of potent neutralizing antibodies, they also present epitopes recognized by non-neutralizing or weakly neutralizing ("off-target") antibodies. Using our recently developed electron microscopy polyclonal epitope mapping approach, we have uncovered a phenomenon wherein off-target antibodies elicited by HIV trimer subunit vaccines cause the otherwise highly stabilized trimeric proteins to degrade into cognate protomers. Further, we show that these protomers expose an expanded suite of off-target epitopes, normally occluded inside the prefusion conformation of trimer, that subsequently elicit further off-target antibody responses. Our study provides critical insights for further improvement of HIV subunit trimer vaccines for future rounds of the iterative vaccine design process.

Correction: Neutralization-guided design of HIV-1 envelope trimers with high affinity for the unmutated common ancestor of CH235 lineage CD4bs broadly neutralizing antibodies.

[This corrects the article DOI: 10.1371/journal.ppat.1008026.].

Neutralization-guided design of HIV-1 envelope trimers with high affinity for the unmutated common ancestor of CH235 lineage CD4bs broadly neutralizing antibodies.

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.

Correction: HIV-1 envelope glycan modifications that permit neutralization by germline-reverted VRC01-class broadly neutralizing antibodies.

[This corrects the article DOI: 10.1371/journal.ppat.1007431.].

HIV-1 envelope glycan modifications that permit neutralization by germline-reverted VRC01-class broadly neutralizing antibodies.

Broadly neutralizing antibody (bnAb) induction is a high priority for effective HIV-1 vaccination. VRC01-class bnAbs that target the CD4 binding site (CD4bs) of trimeric HIV-1 envelope (Env) glycoprotein spikes are particularly attractive to elicit because of their extraordinary breadth and potency of neutralization in vitro and their ability to protect against infection in animal models. Glycans bordering the CD4bs impede the binding of germline-reverted forms of VRC01-class bnAbs and therefore constitute a barrier to early events in initiating the correct antibody lineages. Deleting a subset of these glycans permits Env antigen binding but not virus neutralization, suggesting that additional barriers impede germline-reverted VRC01-class antibody binding to functional Env trimers. We investigated the requirements for functional Env trimer engagement of VRC01-class naïve B cell receptors by using virus neutralization and germline-reverted antibodies as surrogates for the interaction. Targeted deletion of a subset of N-glycans bordering the CD4bs, combined with Man5 enrichment of remaining N-linked glycans that are otherwise processed into larger complex-type glycans, rendered HIV-1 426c Env-pseudotyped virus (subtype C, transmitted/founder) highly susceptible to neutralization by near germline forms of VRC01-class bnAbs. Neither glycan modification alone rendered the virus susceptible to neutralization. The potency of neutralization in some cases rivaled the potency of mature VRC01 against wildtype viruses. Neutralization by the germline-reverted antibodies was abrogated by the known VRC01 resistance mutation, D279K. These findings improve our understanding of the restrictions imposed by glycans in eliciting VRC01-class bnAbs and enable a neutralization-based strategy to monitor vaccine-elicited early precursors of this class of bnAbs.

Structural and immunologic correlates of chemically stabilized HIV-1 envelope glycoproteins.

Inducing broad spectrum neutralizing antibodies against challenging pathogens such as HIV-1 is a major vaccine design goal, but may be hindered by conformational instability within viral envelope glycoproteins (Env). Chemical cross-linking is widely used for vaccine antigen stabilization, but how this process affects structure, antigenicity and immunogenicity is poorly understood and its use remains entirely empirical. We have solved the first cryo-EM structure of a cross-linked vaccine antigen. The 4.2 Å structure of HIV-1 BG505 SOSIP soluble recombinant Env in complex with a CD4 binding site-specific broadly neutralizing antibody (bNAb) Fab fragment reveals how cross-linking affects key properties of the trimer. We observed density corresponding to highly specific glutaraldehyde (GLA) cross-links between gp120 monomers at the trimer apex and between gp120 and gp41 at the trimer interface that had strikingly little impact on overall trimer conformation, but critically enhanced trimer stability and improved Env antigenicity. Cross-links were also observed within gp120 at sites associated with the N241/N289 glycan hole that locally modified trimer antigenicity. In immunogenicity studies, the neutralizing antibody response to cross-linked trimers showed modest but significantly greater breadth against a global panel of difficult-to-neutralize Tier-2 heterologous viruses. Moreover, the specificity of autologous Tier-2 neutralization was modified away from the N241/N289 glycan hole, implying a novel specificity. Finally, we have investigated for the first time T helper cell responses to next-generation soluble trimers, and report on vaccine-relevant immunodominant responses to epitopes within BG505 that are modified by cross-linking. Elucidation of the structural correlates of a cross-linked viral glycoprotein will allow more rational use of this methodology for vaccine design, and reveals a strategy with promise for eliciting neutralizing antibodies needed for an effective HIV-1 vaccine.

Stabilized HIV-1 envelope glycoprotein trimers for vaccine use.

PURPOSE OF REVIEW: To provide an update on the latest developments in the field of HIV-1 antibody-based soluble envelope glycoprotein (Env) trimer design for vaccine use. RECENT FINDINGS: The development of soluble native-like HIV-1 Env trimer immunogens has moved the field of antibody-based vaccine design forward dramatically over the past few years with refinement of various stabilizing approaches. However, despite this progress, significant challenges remain. Firstly, although trimers are relatively stable in solution, they nevertheless sample different conformational states, some of which may be less relevant to binding and induction of broadly neutralizing antibodies (bNAbs). Secondly, these trimers expose unwanted immunodominant surfaces that may distract the adaptive immune response from recognizing more immunorecessive but conserved neutralization-relevant surfaces on the trimer. The availability of atomic-resolution structural information has allowed guided design of mutations that have further stabilized trimers and allowed reduced exposure of unwanted epitopes. Moreover, chemical cross-linking approaches that do not require structural information have also contributed to trimer stabilization and selection of particular conformational forms. However, current knowledge suggests that strategies additional to trimer stabilization will be required to elicit bNAb, including targeting naïve B cell receptors with specific immunogens, and guiding B cell lineages toward recognizing conserved surfaces on Env with high affinity. SUMMARY: This review will give a perspective on these challenges, and summarize current approaches to overcoming them with the aim of developing immunogens to elicit bNAb responses in humans by active vaccination.

Astrocytes Resist HIV-1 Fusion but Engulf Infected Macrophage Material.

HIV-1 disseminates to diverse tissues and establishes long-lived viral reservoirs. These reservoirs include the CNS, in which macrophage-lineage cells, and as suggested by many studies, astrocytes, may be infected. Here, we have investigated astrocyte infection by HIV-1. We confirm that astrocytes trap and internalize HIV-1 particles for subsequent release but find no evidence that these particles infect the cell. Astrocyte infection was not observed by cell-free or cell-to-cell routes using diverse approaches, including luciferase and GFP reporter viruses, fixed and live-cell fusion assays, multispectral flow cytometry, and super-resolution imaging. By contrast, we observed intimate interactions between HIV-1-infected macrophages and astrocytes leading to signals that might be mistaken for astrocyte infection using less stringent approaches. These results have implications for HIV-1 infection of the CNS, viral reservoir formation, and antiretroviral therapy.