Search

Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance., Krebs and colleagues identify multiple mAbs that recognize either the RBD or the NTD of SARS-CoV-2 spike protein that have potent cross-neutralizing activities against variants of concern. Combinatorial mAb cocktails have complementary effects on viral neutralization and Fc effector functions and can protect against SARS-CoV-2 escape mutants.

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a SARS-CoV-2 spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the Alpha (B.1.1.7), and Beta (B.1.351) VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 μg) or low (0.2 μg) dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose vaccinations. Dose-dependent SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and waning immunity call for next-generation vaccine strategies. Here, we assessed the immunogenicity and protective efficacy of two SARS-CoV-2 vaccines targeting the WA1/2020 spike protein, Ad26.COV2.S (Ad26) and Spike ferritin Nanoparticle (SpFN), in nonhuman primates, delivered as either a homologous (SpFN/SpFN and Ad26/Ad26) or heterologous (Ad26/SpFN) prime-boost regimen. The Ad26/SpFN regimen elicited the highest CD4 T cell and memory B cell responses, the SpFN/SpFN regimen generated the highest binding and neutralizing antibody responses, and the Ad26/Ad26 regimen generated the most robust CD8 T cell responses. Despite these differences, protective efficacy against SARS-CoV-2 Omicron BA.1 challenge was similar for all three regimens. After challenge, all vaccinated monkeys showed significantly reduced peak and day 4 viral loads in both bronchoalveolar lavage and nasal swabs as compared with sham animals. The efficacy conferred by these three immunologically distinct vaccine regimens suggests that both humoral and cellular immunity contribute to protection against SARS-CoV-2 Omicron challenge.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019. We developed and evaluated an adjuvanted SARS-CoV-2 spike ferritin nanoparticle (SpFN) vaccine in nonhuman primates. High-dose (50-μg) SpFN vaccine, given twice 28 days apart, induced a T helper cell 1 (TH1)–biased CD4 THresponse and elicited neutralizing antibodies against SARS-CoV-2 wild type and variants of concern, as well as against SARS-CoV-1. These potent humoral and cell-mediated immune responses translated into rapid elimination of replicating virus in the upper and lower airways and lung parenchyma of nonhuman primates after high-dose SARS-CoV-2 respiratory challenge. The immune response elicited by SpFN vaccination and resulting efficacy in nonhuman primates support the utility of SpFN as a vaccine candidate for SARS-causing betacoronaviruses.

HIV-specific chimeric antigen receptor (CAR) T cells that target lymphoid follicles have the potential to functionally cure HIV infection. CD8

The emergence of SARS-CoV-2 variants of concern (VOC) requires adequate coverage of vaccine protection. We evaluated whether a spike ferritin nanoparticle vaccine (SpFN), adjuvanted with the Army Liposomal Formulation QS21 (ALFQ), conferred protection against the B.1.1.7 and B.1.351 VOCs in Syrian golden hamsters. SpFN-ALFQ was administered as either single or double-vaccination (0 and 4 week) regimens, using a high (10 μg) or low (0.2 μg) immunogen dose. Animals were intranasally challenged at week 11. Binding antibody responses were comparable between high- and low-dose groups. Neutralizing antibody titers were equivalent against WA1, B.1.1.7, and B.1.351 variants following two high dose two vaccinations. SpFN-ALFQ vaccination protected against SARS-CoV-2-induced disease and viral replication following intranasal B.1.1.7 or B.1.351 challenge, as evidenced by reduced weight loss, lung pathology, and lung and nasal turbinate viral burden. These data support the development of SpFN-ALFQ as a broadly protective, next-generation SARS-CoV-2 vaccine.

The need for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) next-generation vaccines has been highlighted by the rise of variants of concern (VoCs) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of the prefusion SARS-CoV-2 spike (S), S1, and receptor-binding domain (RBD). These immunogens induce robust S binding, ACE2 inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID50 > 10,000) following a single immunization, whereas RBD-ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations and also show durable and potent neutralization against circulating VoCs. Passive transfer of immunoglobulin G (IgG) purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2-blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens., Graphical abstract, Joyce et al. generate four categories of engineered SARS-CoV-2 ferritin nanoparticle immunogens using structure-based vaccine design that recapitulate the prefusion SARS-CoV-2 spike, S1, and RBD. These immunogens induce robust and protective neutralizing antibody responses against SARS-CoV-2 and elicit potent neutralization against variants of concern and the heterologous SARS-CoV-1.

Significance The emergence of SARS-CoV-2 variants of concern (VOCs) that reduce the efficacy of current COVID-19 vaccines is a major threat to pandemic control. We evaluate a SARS-CoV-2 spike receptor-binding domain ferritin nanoparticle protein vaccine (RFN) in a nonhuman primate challenge model that addresses the need for a next-generation vaccine with increased pan-SARS breadth of coverage. RFN, adjuvanted with a liposomal-QS21 formulation (ALFQ), elicits humoral and cellular immune responses with excellent breadth and potency against SARS-CoV-2 VOCs and SARS-CoV-1, and protects against high-dose respiratory tract challenge with SARS-CoV-2. Our results support consideration of RFN for vaccine development against multiple concerning members of the Sarbecovirus subgenus of Betacoronaviruses., Emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for next-generation vaccines able to elicit broad and durable immunity. Here we report the evaluation of a ferritin nanoparticle vaccine displaying the receptor-binding domain of the SARS-CoV-2 spike protein (RFN) adjuvanted with Army Liposomal Formulation QS-21 (ALFQ). RFN vaccination of macaques using a two-dose regimen resulted in robust, predominantly Th1 CD4+ T cell responses and reciprocal peak mean serum neutralizing antibody titers of 14,000 to 21,000. Rapid control of viral replication was achieved in the upper and lower airways of animals after high-dose SARS-CoV-2 respiratory challenge, with undetectable replication within 4 d in seven of eight animals receiving 50 µg of RFN. Cross-neutralization activity against SARS-CoV-2 variant B.1.351 decreased only approximately twofold relative to WA1/2020. In addition, neutralizing, effector antibody and cellular responses targeted the heterotypic SARS-CoV-1, highlighting the broad immunogenicity of RFN-ALFQ for SARS-CoV−like Sarbecovirus vaccine development.

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants stresses the continued need for next-generation vaccines that confer broad protection against coronavirus disease 2019 (COVID-19). We developed and evaluated an adjuvanted SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine in nonhuman primates (NHPs). High-dose (50µg) SpFN vaccine, given twice within a 28 day interval, induced a Th1-biased CD4 T cell helper response and a peak neutralizing antibody geometric mean titer of 52,773 against wild-type virus, with activity against SARS-CoV-1 and minimal decrement against variants of concern. Vaccinated animals mounted an anamnestic response upon high-dose SARS-CoV-2 respiratory challenge that translated into rapid elimination of replicating virus in their upper and lower airways and lung parenchyma. SpFN’s potent and broad immunogenicity profile and resulting efficacy in NHPs supports its utility as a candidate platform for SARS-like betacoronaviruses.One-Sentence SummaryA SARS-CoV-2 Spike protein ferritin nanoparticle vaccine, co-formulated with a liposomal adjuvant, elicits broad neutralizing antibody responses that exceed those observed for other major vaccines and rapidly protects against respiratory infection and disease in the upper and lower airways and lung tissue of nonhuman primates.

Anti-HIV chimeric antigen receptors (CARs) promote direct killing of infected cells, thus offering a therapeutic approach aimed at durable suppression of infection emerging from viral reservoirs. CD4-based CARs represent a favored option, since they target the essential conserved primary receptor binding site on the HIV envelope glycoprotein (Env). We have previously shown that adding a second Env-binding moiety, such as the carbohydrate recognition domain of human mannose-binding lectin (MBL) that recognizes the highly conserved oligomannose patch on gp120, increases CAR potency in an in vitro HIV suppression assay; moreover it reduces the undesired capacity for the CD4 of the CAR molecule to act as an entry receptor, thereby rendering CAR-expressing CD8+ T cells susceptible to infection. Here, we further improve the bispecific CD4-MBL CAR by adding a third targeting moiety against a distinct conserved Env determinant, i.e. a polypeptide sequence derived from the N-terminus of the HIV coreceptor CCR5. The trispecific CD4-MBL-R5Nt CAR displays enhanced in vitro anti-HIV potency compared to the CD4-MBL CAR, as well as undetectable HIV entry receptor activity. The high anti-HIV potency of the CD4-MBL-R5Nt CAR, coupled with its all-human composition and absence of immunogenic variable regions associated with antibody-based CARs, offer promise for the trispecific construct in therapeutic approaches seeking durable drug-free HIV remission.

SUMMARYSARS-CoV-2 is a zoonotic virus that has caused a pandemic of severe respiratory disease—COVID-19— within several months of its initial identification. Comparable to the first SARS-CoV, this novel coronavirus’s surface Spike (S) glycoprotein mediates cell entry via the human ACE-2 receptor, and, thus, is the principal target for the development of vaccines and immunotherapeutics. Molecular information on the SARS-CoV-2 S glycoprotein remains limited. Here we report the crystal structure of the SARS-CoV-2 S receptor-binding-domain (RBD) at a the highest resolution to date, of 1.95 Å. We identified a set of SARS-reactive monoclonal antibodies with cross-reactivity to SARS-CoV-2 RBD and other betacoronavirus S glycoproteins. One of these antibodies, CR3022, was previously shown to synergize with antibodies that target the ACE-2 binding site on the SARS-CoV RBD and reduce viral escape capacity. We determined the structure of CR3022, in complex with the SARS-CoV-2 RBD, and defined a broadly reactive epitope that is highly conserved across betacoronaviruses. This epitope is inaccessible in the “closed” prefusion S structure, but is accessible in “open” conformations. This first-ever resolution of a human antibody in complex with SARS-CoV-2 and the broad reactivity of this set of antibodies to a conserved betacoronavirus epitope will allow antigenic assessment of vaccine candidates, and provide a framework for accelerated vaccine, immunotherapeutic and diagnostic strategies against SARS-CoV-2 and related betacoronaviruses.HIGHLIGHTSHigh resolution structure of the SARS-CoV-2 Receptor-Binding-Domain (RBD).Recognition of the SARS-CoV-2 RBD by SARS-CoV antibodies.Structure of the SARS-COV-2 RBD in complex with antibody CR3022.Identification of a cryptic site of vulnerability on the SARS-CoV-2 Spike.

Background The zoonotic emergence of SARS-CoV-2 quickly developed into a global pandemic. Multiple vaccine platforms have been advanced to clinical trials and emergency use authorization. The recent emergence of SARS-CoV-2 virus variants with Spike receptor-binding domain (RBD) and N-terminal domain (NTD) mutations, highlights the need for next-generation vaccines that can elicit immune responses that are resilient against Spike mutations. Methods Using a structure-based vaccine design approach, we developed multiple optimized SARS-CoV-2 nanoparticle immunogens that recapitulate the structural and antigenic profile of the SARS-CoV-2 prefusion spike. We assessed these immunogens in murine immunogenicity studies and in a K18-hACE2 transgenic mouse model with a SARS-CoV-2 challenge. Immune sera from vaccinated mice were assessed for SARS-CoV-2 binding, and neutralization against SARS-CoV-2, variants of concern, and the heterologous SARS-CoV-1 virus. Results In combination with a liposomal-saponin based adjuvant (ALFQ), these immunogens induced robust binding, ACE2-inhibition, and authentic virus and pseudovirus neutralization. A Spike-Ferritin nanoparticle (SpFN) vaccine elicited neutralizing ID50 titers >10,000 after a single immunization, while RBD-Ferritin (RFN) nanoparticle immunogens elicited ID50 titer values >10,000 values after two immunizations. Purified antibody from SpFN- or RFN-immunized mice was transfused into K18-ACE2 transgenic mice and challenged with a high-dose SARS-CoV-2 virus stock. In order to understand the breadth of vaccine-elicited antibody responses, we analyzed SpFN- and RBD-FN-immunized animal sera against a set of heterologous SARS-CoV-2 RBD variants and SARS-CoV RBD. High binding titers with ACE2-blocking activity were observed against SARS-CoV-2 variants and the heterologous SARS-CoV-1 RBD. Furthermore, both SpFN- and RFN-immunized animal sera showed SARS-CoV-1 neutralizing ID50 titers of >2000. Conclusion These observations highlight the importance of SARS-CoV-2 neutralizing antibody levels in providing protection against emerging SARS-like coronaviruses and provide a robust platform for pandemic preparedness. Structure-based design enables development of a SARS-CoV-2 nanoparticle immunogen. Disclosures All Authors: No reported disclosures

There is a need to develop improved methods to treat and potentially cure HIV infection. During chronic HIV infection, replication is concentrated within T follicular helper cells (Tfh) located within B cell follicles, where low levels of virus-specific CTL permit ongoing viral replication. We previously showed that elevated levels of simian immunodeficiency virus (SIV)-specific CTL in B cell follicles are linked to both decreased levels of viral replication in follicles and decreased plasma viral loads. These findings provide the rationale to develop a strategy for targeting follicular viral-producing (Tfh) cells using antiviral chimeric antigen receptor (CAR) T cells co-expressing the follicular homing chemokine receptor CXCR5. We hypothesize that antiviral CAR/CXCR5-expressing T cells, when infused into an SIV-infected animal or an HIV-infected individual, will home to B cell follicles, suppress viral replication, and lead to long-term durable remission of SIV and HIV. To begin to test this hypothesis, we engineered gammaretroviral transduction vectors for co-expression of a bispecific anti-SIV CAR and rhesus macaque CXCR5. Viral suppression by CAR/CXCR5-transduced T cells was measured in vitro, and CXCR5-mediated migration was evaluated using both an in vitro transwell migration assay, as well as a novel ex vivo tissue migration assay. The functionality of the CAR/CXCR5 T cells was demonstrated through their potent suppression of SIVmac239 and SIVE660 replication in in vitro and migration to the ligand CXCL13 in vitro, and concentration in B cell follicles in tissues ex vivo. These novel antiviral immunotherapy products have the potential to provide long-term durable remission (functional cure) of HIV and SIV infections.

Background aims Chimeric antigen receptors (CARs) offer great potential toward a functional cure of human immunodeficiency virus (HIV) infection. To achieve the necessary long-term virus suppression, we believe that CARs must be designed for optimal potency and anti-HIV specificity, and also for minimal probability of virus escape and CAR immunogenicity. CARs containing antibody-based motifs are problematic in the latter regard due to epitope mutation and anti-idiotypic immune responses against the variable regions. Methods We designed bispecific CARs, each containing a segment of human CD4 linked to the carbohydrate recognition domain of a human C-type lectin. These CARs target two independent regions on HIV-1 gp120 that presumably must be conserved on clinically significant virus variants (i.e., the primary receptor binding site and the dense oligomannose patch). Functionality and specificity of these bispecific CARs were analyzed in assays of CAR-T cell activation and spreading HIV-1 suppression. Results T cells expressing a CD4-dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DCSIGN) CAR displayed robust stimulation upon encounter with Env-expressing targets, but negligible activity against intercellular adhesion molecule (ICAM)-2 and ICAM-3, the natural dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin ligands. Moreover, the presence of the lectin moiety prevented the CD4 from acting as an entry receptor on CCR5-expressing cells, including CD8+ T cells. However, in HIV suppression assays, the CD4-DCSIGN CAR and the related CD4-liver/lymph node-specific intercellular adhesion molecule-3-grabbing non-integrin CAR displayed only minimally increased potency compared with the CD4 CAR against some HIV-1 isolates and reduced potency against others. By contrast, the CD4-langerin and CD4-mannose binding lectin (MBL) CARs uniformly displayed enhanced potency compared with the CD4 CAR against all the genetically diverse HIV-1 isolates examined. Further experimental data, coupled with known biological features, suggest particular advantages of the CD4-MBL CAR. Discussion These studies highlight features of bispecific CD4-lectin CARs that achieve potency enhancement by targeting two distinct highly conserved Env determinants while lacking immunogenicity-prone antibody-based motifs.

Membrane proteins comprise a third of the human genome, yet present challenging targets for reverse chemical genetics. For example, although implicated in numerous diseases including multiple myeloma, the membrane protein caveolin-1 appears to offer a poor target for the discovery of synthetic ligands due to its largely unknown structure and insolubility. To break this impasse and identify new classes of caveolae controlling lead compounds, we applied phage-based, reverse chemical genetics for the discovery of caveolin-1 ligands derived from the anti-HIV therapeutic T20. Substitution of homologous residues into the T20 sequence used a process analogous to medicinal chemistry for the affinity maturation to bind caveolin. The resultant caveolin-1 ligands bound with1000-fold higher affinity than wild-type T20. Two types of ELISAs and isothermal titration calorimetry (ITC) measurements demonstrated high affinity binding to caveolin by the T20 variants with K(d) values in the 150 nM range. Microscopy experiments with the highest affinity caveolin ligands confirmed colocalization of the ligands with endogenous caveolin in NIH 3T3 cells. The results establish the foundation for targeting caveolin and caveolae formation in living cells.

Chimeric antigen receptor (CAR) technology has potential to achieve durable (life-long?) control of HIV in the absence of antiretroviral drugs, i.e. a “functional cure”. Thus in targeting the HIV-1 Env glycoprotein expressed on the surface of virus-producing cells, CAR design must strive not only for high potency and HIV specificity, but also to approach the ideal qualities of inescapablilty and non-immunogenicity. Our strategy involves bispecific targeting domains consisting of an extracellular region of human CD4, the primary HIV receptor, attached to a second motif that recognizes a distinct highly conserved region on the otherwise highly variable Env. The second motif is intended both to enhance CAR potency, and to prevent the CD4 from acting as an HIV entry receptor on CAR-expressing CD8+ T cells. We have focused on second motifs predicted to lack the immunogenicity associated with antibody-based CARs. A particularly favorable construct contains CD4 linked to the carbohydrate recognition domain of human mannose-binding lectin (MBL), which recognizes the highly conserved oligomannose patch on gp120 (Ghanem et al., Cytotherapy). In another variant, CD4 is linked to the N-terminal portion of the CCR5 co-receptor. We found that the bispecific CARs broadly suppressed infection by genetically diverse HIV-1 isolates, with significantly greater potency than a monospecifc CD4 CAR. Similar results were obtained using rhesus macaque PBMCs and a rhesus version of the CD4-MBL CAR. These results set the stage for testing the activities of the CD4-based bispecific CARs in relevant animal models including rhesus macaques and humanized mice.

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intra-membrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin, an experiment requiring purified proteins. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intra-membrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

Phage display of protein and peptide libraries offers a powerful technology for the selection and isolation of ligands and receptors. To date, the technique has been considered limited to soluble, non-membrane proteins. We report two examples of phage display of full-length, folded and functional membrane proteins. Consistent display required the recently reported KO7(+) helper phage. The two proteins, full-length caveolin-1 and HIV gp41, display well on the surface of the phage, and maintain their binding activities as shown by in vitro assays.

During HIV entry, the surface-exposed gp120 subunit of the envelope glycoprotein (Env) undergoes a series of stepwise conformational changes in response to interactions with the primary receptor CD4, and co-receptors, CCR5 or CXCR4. Obtaining structural information on the various intermediates during viral entry is a key focus of antiviral and vaccine research and could open the doors for more effective treatment and prevention. This project aims to develop recombinant soluble co-receptor mimetics featuring critical determinants of the chemokine receptor, CCR5, using two approaches: fusing the CCR5 moieties to soluble CD4 by flexible polypeptide linkers and using a globular, stable scaffold protein to orient the co-receptor determinants in an optimal conformation to recreate the binding interaction with gp120. We have successfully overexpressed and purified the recombinant proteins from mammalian cells. Characterization of the gp120-binding properties of the variants is underway using a vaccinia-based cell fusion assay where soluble CD4 has been shown to induce membrane fusion between Env-expressing effector cells and target cells bearing CCR5, but no CD4. The CCR5 moieties of the sCD4-fused constructs compete with cell-surface CCR5 for binding to gp120, leading to impaired activation of membrane fusion compared with sCD4. The soluble variants of CCR5 will be used in experiments to elucidate the conformational changes in gp120 that immediately precede membrane fusion.