Subcutaneous Administration of a Monoclonal Antibody to Prevent Malaria.

BACKGROUND: Subcutaneous administration of the monoclonal antibody L9LS protected adults against controlled Plasmodium falciparum infection in a phase 1 trial. Whether a monoclonal antibody administered subcutaneously can protect children from P. falciparum infection in a region where this organism is endemic is unclear. METHODS: We conducted a phase 2 trial in Mali to assess the safety and efficacy of subcutaneous administration of L9LS in children 6 to 10 years of age over a 6-month malaria season. In part A of the trial, safety was assessed at three dose levels in adults, followed by assessment at two dose levels in children. In part B of the trial, children were randomly assigned, in a 1:1:1 ratio, to receive 150 mg of L9LS, 300 mg of L9LS, or placebo. The primary efficacy end point, assessed in a time-to-event analysis, was the first P. falciparum infection, as detected on blood smear performed at least every 2 weeks for 24 weeks. A secondary efficacy end point was the first episode of clinical malaria, as assessed in a time-to-event analysis. RESULTS: No safety concerns were identified in the dose-escalation part of the trial (part A). In part B, 225 children underwent randomization, with 75 children assigned to each group. No safety concerns were identified in part B. P. falciparum infection occurred in 36 participants (48%) in the 150-mg group, in 30 (40%) in the 300-mg group, and in 61 (81%) in the placebo group. The efficacy of L9LS against P. falciparum infection, as compared with placebo, was 66% (adjusted confidence interval [95% CI], 45 to 79) with the 150-mg dose and 70% (adjusted 95% CI, 50 to 82) with the 300-mg dose (P<0.001 for both comparisons). Efficacy against clinical malaria was 67% (adjusted 95% CI, 39 to 82) with the 150-mg dose and 77% (adjusted 95% CI, 55 to 89) with the 300-mg dose (P<0.001 for both comparisons). CONCLUSIONS: Subcutaneous administration of L9LS to children was protective against P. falciparum infection and clinical malaria over a period of 6 months. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT05304611.).

NK cell-induced damage to P.falciparum-infected erythrocytes requires ligand-specific recognition and releases parasitophorous vacuoles that are phagocytosed by monocytes in the presence of immune IgG.

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin αMß2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P. falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P.falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK-iRBC synapses and free vesicles similar in size to GFP+ PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P.falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P. falciparum infection.

Creation of a non-Western humanized gnotobiotic mouse model through the transplantation of rural African fecal microbiota.

IMPORTANCE: There is increasing evidence that microbes residing within the intestines (gut microbiota) play important roles in the well-being of humans. Yet, there are considerable challenges in determining the specific role of gut microbiota in human diseases owing to the complexity of diverse internal and environmental factors that can contribute to diseases. Mice devoid of all microorganisms (germ-free mice) can be colonized with human stool samples to examine the specific contribution of the gut microbiota to a disease. These approaches have been primarily focused on stool samples obtained from individuals in Western countries. Thus, there is limited understanding as to whether the same methods used to colonize germ-free mice with stool from Western individuals would apply to the colonization of germ-free mice with stool from non-Western individuals. Here, we report the results from colonizing germ-free mice with stool samples of Malian children.

Accumulation of Neutrophil Phagocytic Antibody Features Tracks With Naturally Acquired Immunity Against Malaria in Children.

BACKGROUND: Studies have demonstrated the protective role of antibodies against malaria. Young children are known to be particularly vulnerable to malaria, pointing to the evolution of naturally acquired clinical immunity over time. However, whether changes in antibody functionality track with the acquisition of naturally acquired malaria immunity remains incompletely understood. METHODS: Using systems serology, we characterized sporozoite- and merozoite-specific antibody profiles of uninfected Malian children before the malaria season who differed in their ability to control parasitemia and fever following Plasmodium falciparum (Pf) infection. We then assessed the contributions of individual traits to overall clinical outcomes, focusing on the immunodominant sporozoite CSP and merozoite AMA1 and MSP1 antigens. RESULTS: Humoral immunity evolved with age, with an expansion of both magnitude and functional quality, particularly within blood-stage phagocytic antibody activity. Moreover, concerning clinical outcomes postinfection, protected children had higher antibody-dependent neutrophil activity along with higher levels of MSP1-specific IgG3 and IgA and CSP-specific IgG3 and IgG4 prior to the malaria season. CONCLUSIONS: These data point to the natural evolution of functional humoral immunity to Pf with age and highlight particular antibody Fc-effector profiles associated with the control of malaria in children, providing clues for the design of next-generation vaccines or therapeutics.

Neither the African-Centric S47 Nor P72 Variant of TP53 Is Associated With Reduced Risk of Febrile Malaria in a Malian Cohort Study.

BACKGROUND: TP53 has been shown to play a role in inflammatory processes, including malaria. We previously found that p53 attenuates parasite-induced inflammation and predicts clinical protection to Plasmodium falciparum infection in Malian children. Here, we investigated whether p53 codon 47 and 72 polymorphisms are associated with differential risk of P. falciparum infection and uncomplicated malaria in a prospective cohort study of malaria immunity. METHODS: p53 codon 47 and 72 polymorphisms were determined by sequencing TP53 exon 4 in 631 Malian children and adults enrolled in the Kalifabougou cohort study. The effects of these polymorphisms on the prospective risk of febrile malaria, incident parasitemia, and time to fever after incident parasitemia over 6 months of intense malaria transmission were assessed using Cox proportional hazards models. RESULTS: Confounders of malaria risk, including age and hemoglobin S or C, were similar between individuals with or without p53 S47 and R72 polymorphisms. Relative to their respective common variants, neither S47 nor R72 was associated with differences in prospective risk of febrile malaria, incident parasitemia, or febrile malaria after parasitemia. CONCLUSIONS: These findings indicate that p53 codon 47 and 72 polymorphisms are not associated with protection against incident P. falciparum parasitemia or uncomplicated febrile malaria.

Evidence for exposure dependent carriage of malaria parasites across the dry season: modelling analysis of longitudinal data.

BACKGROUND: In malaria endemic regions, transmission of Plasmodium falciparum parasites is often seasonal with very low transmission during the dry season and high transmission in the wet season. Parasites survive the dry season within some individuals who experience prolonged carriage of parasites and are thought to 'seed' infection in the next transmission season. METHODS: Dry season carriers and their role in the subsequent transmission season are characterized using a combination of mathematical simulations and data analysis of previously described data from a longitudinal study in Mali of individuals aged 3 months-12 years (n = 579). RESULTS: Simulating the life-history of individuals experiencing repeated exposure to infection predicts that dry season carriage is more likely in the oldest, most exposed and most immune individuals. This hypothesis is supported by the data from Mali, which shows that carriers are significantly older, experience a higher biting rate at the beginning of the transmission season and develop clinical malaria later than non-carriers. Further, since the most exposed individuals in a community are most likely to be dry season carriers, this is predicted to enable a more than twofold faster spread of parasites into the mosquito population at the start of the subsequent wet season. CONCLUSIONS: Carriage of malaria parasites over the months-long dry season in Mali is most likely in the older, more exposed and more immune children. These children may act as super-spreaders facilitating the fast spread of parasites at the beginning of the next transmission season.

Bivalent intra-spike binding provides durability against emergent Omicron lineages: Results from a global consortium.

The SARS-CoV-2 Omicron variant of concern (VoC) and its sublineages contain 31-36 mutations in spike and escape neutralization by most therapeutic antibodies. In a pseudovirus neutralization assay, 66 of the nearly 400 candidate therapeutics in the Coronavirus Immunotherapeutic Consortium (CoVIC) panel neutralize Omicron and multiple Omicron sublineages. Among natural immunoglobulin Gs (IgGs), especially those in the receptor-binding domain (RBD)-2 epitope community, nearly all Omicron neutralizers recognize spike bivalently, with both antigen-binding fragments (Fabs) simultaneously engaging adjacent RBDs on the same spike. Most IgGs that do not neutralize Omicron bind either entirely monovalently or have some (22%-50%) monovalent occupancy. Cleavage of bivalent-binding IgGs to Fabs abolishes neutralization and binding affinity, with disproportionate loss of activity against Omicron pseudovirus and spike. These results suggest that VoC-resistant antibodies overcome mutagenic substitution via avidity. Hence, vaccine strategies targeting future SARS-CoV-2 variants should consider epitope display with spacing and organization identical to trimeric spike.

Rare, convergent antibodies targeting the stem helix broadly neutralize diverse betacoronaviruses.

Humanity has faced three recent outbreaks of novel betacoronaviruses, emphasizing the need to develop approaches that broadly target coronaviruses. Here, we identify 55 monoclonal antibodies from COVID-19 convalescent donors that bind diverse betacoronavirus spike proteins. Most antibodies targeted an S2 epitope that included the K814 residue and were non-neutralizing. However, 11 antibodies targeting the stem helix neutralized betacoronaviruses from different lineages. Eight antibodies in this group, including the six broadest and most potent neutralizers, were encoded by IGHV1-46 and IGKV3-20. Crystal structures of three antibodies of this class at 1.5-1.75-Å resolution revealed a conserved mode of binding. COV89-22 neutralized SARS-CoV-2 variants of concern including Omicron BA.4/5 and limited disease in Syrian hamsters. Collectively, these findings identify a class of IGHV1-46/IGKV3-20 antibodies that broadly neutralize betacoronaviruses by targeting the stem helix but indicate these antibodies constitute a small fraction of the broadly reactive antibody response to betacoronaviruses after SARS-CoV-2 infection.

Application of B cell immortalization for the isolation of antibodies and B cell clones from vaccine and infection settings.

The isolation and characterization of neutralizing antibodies from infection and vaccine settings informs future vaccine design, and methodologies that streamline the isolation of antibodies and the generation of B cell clones are of great interest. Retroviral transduction to express Bcl-6 and Bcl-xL and transform primary B cells has been shown to promote long-term B cell survival and antibody secretion in vitro, and can be used to isolate antibodies from memory B cells. However, application of this methodology to B cell subsets from different tissues and B cells from chronically infected individuals has not been well characterized. Here, we characterize Bcl-6/Bcl-xL B cell immortalization across multiple tissue types and B cell subsets in healthy and HIV-1 infected individuals, as well as individuals recovering from malaria. In healthy individuals, naïve and memory B cell subsets from PBMCs and tonsil tissue transformed with similar efficiencies, and displayed similar characteristics with respect to their longevity and immunoglobulin secretion. In HIV-1-viremic individuals or in individuals with recent malaria infections, the exhausted CD27-CD21- memory B cells transformed with lower efficiency, but the transformed B cells expanded and secreted IgG with similar efficiency. Importantly, we show that this methodology can be used to isolate broadly neutralizing antibodies from HIV-infected individuals. Overall, we demonstrate that Bcl-6/Bcl-xL B cell immortalization can be used to isolate antibodies and generate B cell clones from different B cell populations, albeit with varying efficiencies.

Safety and Efficacy of a Monoclonal Antibody against Malaria in Mali.

BACKGROUND: CIS43LS is a monoclonal antibody that was shown to protect against controlled Plasmodium falciparum infection in a phase 1 clinical trial. Whether a monoclonal antibody can prevent P. falciparum infection in a region in which the infection is endemic is unknown. METHODS: We conducted a phase 2 trial to assess the safety and efficacy of a single intravenous infusion of CIS43LS against P. falciparum infection in healthy adults in Mali over a 6-month malaria season. In Part A, safety was assessed at three escalating dose levels. In Part B, participants were randomly assigned (in a 1:1:1 ratio) to receive 10 mg of CIS43LS per kilogram of body weight, 40 mg of CIS43LS per kilogram, or placebo. The primary efficacy end point, assessed in a time-to-event analysis, was the first P. falciparum infection detected on blood-smear examination, which was performed at least every 2 weeks for 24 weeks. At enrollment, all the participants received artemether-lumefantrine to clear possible P. falciparum infection. RESULTS: In Part B, 330 adults underwent randomization; 110 were assigned to each trial group. The risk of moderate headache was 3.3 times as high with 40 mg of CIS43LS per kilogram as with placebo. P. falciparum infections were detected on blood-smear examination in 39 participants (35.5%) who received 10 mg of CIS43LS per kilogram, 20 (18.2%) who received 40 mg of CIS43LS per kilogram, and 86 (78.2%) who received placebo. At 6 months, the efficacy of 40 mg of CIS43LS per kilogram as compared with placebo was 88.2% (adjusted 95% confidence interval [CI], 79.3 to 93.3; P<0.001), and the efficacy of 10 mg of CIS43LS per kilogram as compared with placebo was 75.0% (adjusted 95% CI, 61.0 to 84.0; P<0.001). CONCLUSIONS: CIS43LS was protective against P. falciparum infection over a 6-month malaria season in Mali without evident safety concerns. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT04329104.).

Neutralizing and interfering human antibodies define the structural and mechanistic basis for antigenic diversion.

Defining mechanisms of pathogen immune evasion and neutralization are critical to develop potent vaccines and therapies. Merozoite Surface Protein 1 (MSP-1) is a malaria vaccine antigen and antibodies to MSP-1 are associated with protection from disease. However, MSP-1-based vaccines performed poorly in clinical trials in part due to a limited understanding of the protective antibody response to MSP-1 and of immune evasion by antigenic diversion. Antigenic diversion was identified as a mechanism wherein parasite neutralization by a MSP-1-specific rodent antibody was disrupted by MSP-1-specific non-inhibitory blocking/interfering antibodies. Here, we investigated a panel of MSP-1-specific naturally acquired human monoclonal antibodies (hmAbs). Structures of multiple hmAbs with diverse neutralizing potential in complex with MSP-1 revealed the epitope of a potent strain-transcending hmAb. This neutralizing epitope overlaps with the epitopes of high-affinity non-neutralizing hmAbs. Strikingly, the non-neutralizing hmAbs outcompete the neutralizing hmAb enabling parasite survival. These findings demonstrate the structural and mechanistic basis for a generalizable pathogen immune evasion mechanism through neutralizing and interfering human antibodies elicited by antigenic diversion, and provides insights required to develop potent and durable malaria interventions.

Broadly neutralizing antibodies target the coronavirus fusion peptide.

The potential for future coronavirus outbreaks highlights the need to broadly target this group of pathogens. We used an epitope-agnostic approach to identify six monoclonal antibodies that bind to spike proteins from all seven human-infecting coronaviruses. All six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. COV44-62 and COV44-79 broadly neutralize alpha- and betacoronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants BA.2 and BA.4/5, albeit with lower potency than receptor binding domain-specific antibodies. In crystal structures of COV44-62 and COV44-79 antigen-binding fragments with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine residue at the S2' cleavage site. COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings highlight the fusion peptide as a candidate epitope for next-generation coronavirus vaccine development.

Atypical B cells up-regulate costimulatory molecules during malaria and secrete antibodies with T follicular helper cell support.

Several infectious and autoimmune diseases are associated with an expansion of CD21-CD27- atypical B cells (atBCs) that up-regulate inhibitory receptors and exhibit altered B cell receptor (BCR) signaling. The function of atBCs remains unclear, and few studies have investigated the biology of pathogen-specific atBCs during acute infection. Here, we performed longitudinal flow cytometry analyses and RNA sequencing of Plasmodium falciparum (Pf)-specific B cells isolated from study participants before and shortly after febrile malaria, with simultaneous analysis of influenza hemagglutinin (HA)-specific B cells as a comparator. At the healthy baseline before the malaria season, individuals had similar frequencies of Pf- and HA-specific atBCs that did not differ proportionally from atBCs within the total B cell population. BCR sequencing identified clonal relationships between Pf-specific atBCs, activated B cells (actBCs), and classical memory B cells (MBCs) and revealed comparable degrees of somatic hypermutation. At the healthy baseline, Pf-specific atBCs were transcriptionally distinct from Pf-specific actBCs and classical MBCs. In response to acute febrile malaria, Pf-specific atBCs and actBCs up-regulated similar intracellular signaling cascades. Pf-specific atBCs showed activation of pathways involved in differentiation into antibody-secreting cells and up-regulation of molecules that mediate B-T cell interactions, suggesting that atBCs respond to T follicular helper (TFH) cells. In the presence of TFH cells and staphylococcal enterotoxin B, atBCs of malaria-exposed individuals differentiated into CD38+ antibody-secreting cells in vitro, suggesting that atBCs may actively contribute to humoral immunity to infectious pathogens.

Broadly neutralizing antibodies target the coronavirus fusion peptide.

The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2' cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2' cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development. One-Sentence Summary: Rare monoclonal antibodies from COVID-19 convalescent individuals broadly neutralize coronaviruses by targeting the fusion peptide.

Design and implementation of multiplexed amplicon sequencing panels to serve genomic epidemiology of infectious disease: A malaria case study.

Multiplexed PCR amplicon sequencing (AmpSeq) is an increasingly popular application for cost-effective monitoring of threatened species and managed wildlife populations, and shows strong potential for the genomic epidemiology of infectious disease. AmpSeq data from infectious microbes can inform disease control in multiple ways, such as by measuring drug resistance marker prevalence, distinguishing imported from local cases, and determining the effectiveness of therapeutics. We describe the design and comparative evaluation of two new AmpSeq assays for Plasmodium falciparum malaria parasites: a four-locus panel ("4CAST") composed of highly diverse antigens, and a 129-locus panel ("AMPLseq") composed of drug resistance markers, highly diverse loci for inferring relatedness, and a locus to detect Plasmodium vivax co-infection. We explore the performance of each panel in various public health use cases with in silico simulations as well as empirical experiments. The 4CAST panel appears highly suitable for evaluating the number of distinct parasite strains within samples (complexity of infection), showing strong performance across a wide range of parasitaemia levels without a DNA pre-amplification step. For relatedness inference, the larger AMPLseq panel performs similarly to two existing panels of comparable size, despite differences in the data and approach used for designing each panel. Finally, we describe an R package (paneljudge) that facilitates the design and comparative evaluation of genetic panels for relatedness estimation, and we provide general guidance on the design and implementation of AmpSeq panels for the genomic epidemiology of infectious disease.

Repeated Plasmodium falciparum infection in humans drives the clonal expansion of an adaptive γδ T cell repertoire.

Repeated Plasmodium falciparum infections drive the development of clinical immunity to malaria in humans; however, the immunological mechanisms that underpin this response are only partially understood. We investigated the impact of repeated P. falciparum infections on human γδ T cells in the context of natural infection in Malian children and adults, as well as serial controlled human malaria infection (CHMI) of U.S. adults, some of whom became clinically immune to malaria. In contrast to the predominant Vδ2+ T cell population in malaria-naïve Australian individuals, clonally expanded cytotoxic Vδ1effector T cells were enriched in the γδ T cell compartment of Malian subjects. Malaria-naïve U.S. adults exposed to four sequential CHMIs defined the precise impact of P. falciparum on the γδ T cell repertoire. Specifically, innate-like Vδ2+ T cells exhibited an initial robust polyclonal response to P. falciparum infection that was not sustained with repeated infections, whereas Vδ1+ T cells increased in frequency with repeated infections. Moreover, repeated P. falciparum infection drove waves of clonal selection in the Vδ1+ T cell receptor repertoire that coincided with the differentiation of Vδ1naïve T cells into cytotoxic Vδ1effector T cells. Vδ1+ T cells of malaria-exposed Malian and U.S. individuals were licensed for reactivity to P. falciparum parasites in vitro. Together, our study indicates that repeated P. falciparum infection drives the clonal expansion of an adaptive γδ T cell repertoire and establishes a role for Vδ1+ T cells in the human immune response to malaria.

A genotyping assay to determine geographic origin and transmission potential of Plasmodium falciparum malaria cases.

As countries work towards malaria elimination, it is important to monitor imported cases to prevent reestablishment of local transmission. The Plasmodium falciparum Pfs47 gene has strong geographic population structure, because only those parasites with Pfs47 haplotypes compatible with the mosquito vector species in a given continent are efficiently transmitted. Analysis of 4,971 world-wide Pfs47 sequences identified two SNPs (at 707 and 725 bp) as sufficient to establish the likely continent of origin of P. falciparum isolates. Pfs47 sequences from Africa, Asia, and the New World presented more that 99% frequency of distinct combinations of the SNPs 707 and 725 genotypes. Interestingly, Papua New Guinea Pfs47 sequences have the highest diversity in SNPs 707 and 725. Accurate and reproducible High-Resolution Melting (HRM) assays were developed to genotype Pfs47 SNPs 707 and 725 in laboratory and field samples, to assess the geographic origin and risk of local transmission of imported P. falciparum malaria cases.

Bispecific antibodies targeting distinct regions of the spike protein potently neutralize SARS-CoV-2 variants of concern.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of existing vaccines and therapeutic antibodies and underscores the need for additional antibody-based tools that potently neutralize variants by targeting multiple sites of the spike protein. We isolated 216 monoclonal antibodies targeting SARS-CoV-2 from plasmablasts and memory B cells collected from patients with coronavirus disease 2019. The three most potent antibodies targeted distinct regions of the receptor binding domain (RBD), and all three neutralized the SARS-CoV-2 Alpha and Beta variants. The crystal structure of the most potent antibody, CV503, revealed that it binds to the ridge region of SARS-CoV-2 RBD, competes with the angiotensin-converting enzyme 2 receptor, and has limited contact with key variant residues K417, E484, and N501. We designed bispecific antibodies by combining nonoverlapping specificities and identified five bispecific antibodies that inhibit SARS-CoV-2 infection at concentrations of less than 1 ng/ml. Through a distinct mode of action, three bispecific antibodies cross-linked adjacent spike proteins using dual N-terminal domain­RBD specificities. One bispecific antibody was greater than 100-fold more potent than a cocktail of its parent monoclonals in vitro and prevented clinical disease in a hamster model at a dose of 2.5 mg/kg. Two bispecific antibodies in our panel comparably neutralized the Alpha, Beta, Gamma, and Delta variants and wild-type virus. Furthermore, a bispecific antibody that neutralized the Beta variant protected hamsters against SARS-CoV-2 expressing the E484K mutation. Thus, bispecific antibodies represent a promising next-generation countermeasure against SARS-CoV-2 variants of concern.