Generation and Study of Antibodies against Two Triangular Trimers Derived from Aß.

Monoclonal antibodies (mAbs) that target the P-amyloid peptide (Aß) are important Alzheimer's disease research tools and are now being used as Alzheimer's disease therapies. Conformation-specific mAbs that target oligomeric and fibrillar Aß assemblies are of particular interest, as these assemblies are associated with Alzheimer's disease pathogenesis and progression. This paper reports the generation of rabbit mAbs against two different triangular trimers derived from Aß. These antibodies are the first mAbs generated against Aß oligomer mimics in which the high-resolution structures of the oligomers are known. We describe the isolation of the mAbs using single B-cell sorting of peripheral blood mononuclear cells (PBMCs) from immunized rabbits, the selectivity of the mAbs for the triangular trimers, the immunoreactivity of the mAbs with aggregated Aß42, and the immunoreactivity of the mAbs in brain tissue from the 5xFAD Alzheimer's disease mouse model. The characterization of these mAbs against structurally defined trimers derived from Aß enhances understanding of antibody-amyloid recognition and may benefit the development of diagnostics and immunotherapies in Alzheimer's disease.

Probing differences among Aß oligomers with two triangular trimers derived from Aß.

The assembly of the ß-amyloid peptide (Aß) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aß is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aß oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aß. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aß, with one trimer showing a greater propensity to interact with Aß than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aß. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aß trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aß oligomers.

A Powassan virus domain III nanoparticle immunogen elicits neutralizing and protective antibodies in mice.

Powassan virus (POWV) is an emerging tick borne flavivirus (TBFV) that causes severe neuroinvasive disease. Currently, there are no approved treatments or vaccines to combat POWV infection. Here, we generated and characterized a nanoparticle immunogen displaying domain III (EDIII) of the POWV E glycoprotein. Immunization with POWV EDIII presented on nanoparticles resulted in significantly higher serum neutralizing titers against POWV than immunization with monomeric POWV EDIII. Furthermore, passive transfer of EDIII-reactive sera protected against POWV challenge in vivo. We isolated and characterized a panel of EDIII-specific monoclonal antibodies (mAbs) and identified several that potently inhibit POWV infection and engage distinct epitopes within the lateral ridge and C-C' loop of the EDIII. By creating a subunit-based nanoparticle immunogen with vaccine potential that elicits antibodies with protective activity against POWV infection, our findings enhance our understanding of the molecular determinants of antibody-mediated neutralization of TBFVs.

Resurfaced ZIKV EDIII nanoparticle immunogens elicit neutralizing and protective responses in vivo.

Zika virus (ZIKV) is a flavivirus that can cause severe disease, but there are no approved treatments or vaccines. A complication for flavivirus vaccine development is the potential of immunogens to enhance infection via antibody-dependent enhancement (ADE), a process mediated by poorly neutralizing and cross-reactive antibodies. Thus, there is a great need to develop immunogens that minimize the potential to elicit enhancing antibodies. Here we utilized structure-based protein engineering to develop "resurfaced" (rs) ZIKV immunogens based on E glycoprotein domain III (ZDIIIs), in which epitopes bound by variably neutralizing antibodies were masked by combinatorial mutagenesis. We identified one resurfaced ZDIII immunogen (rsZDIII-2.39) that elicited a protective but immune-focused response. Compared to wild type ZDIII, immunization with resurfaced rsZDIII-2.39 protein nanoparticles produced fewer numbers of ZIKV EDIII antigen-reactive B cells and elicited serum that had a lower magnitude of induced ADE against dengue virus serotype 1 (DENV1) Our findings enhance our understanding of the structural and functional determinants of antibody protection against ZIKV.

Longitudinally monitored immune biomarkers predict the timing of COVID-19 outcomes.

The clinical outcome of SARS-CoV-2 infection varies widely between individuals. Machine learning models can support decision making in healthcare by assessing fatality risk in patients that do not yet show severe signs of COVID-19. Most predictive models rely on static demographic features and clinical values obtained upon hospitalization. However, time-dependent biomarkers associated with COVID-19 severity, such as antibody titers, can substantially contribute to the development of more accurate outcome models. Here we show that models trained on immune biomarkers, longitudinally monitored throughout hospitalization, predicted mortality and were more accurate than models based on demographic and clinical data upon hospital admission. Our best-performing predictive models were based on the temporal analysis of anti-SARS-CoV-2 Spike IgG titers, white blood cell (WBC), neutrophil and lymphocyte counts. These biomarkers, together with C-reactive protein and blood urea nitrogen levels, were found to correlate with severity of disease and mortality in a time-dependent manner. Shapley additive explanations of our model revealed the higher predictive value of day post-symptom onset (PSO) as hospitalization progresses and showed how immune biomarkers contribute to predict mortality. In sum, we demonstrate that the kinetics of immune biomarkers can inform clinical models to serve as a powerful monitoring tool for predicting fatality risk in hospitalized COVID-19 patients, underscoring the importance of contextualizing clinical parameters according to their time post-symptom onset.

Monoclonal antibodies from humans with Mycobacterium tuberculosis exposure or latent infection recognize distinct arabinomannan epitopes.

The surface polysacharide arabinomannan (AM) and related glycolipid lipoarabinomannan (LAM) play critical roles in tuberculosis pathogenesis. Human antibody responses to AM/LAM are heterogenous and knowledge of reactivity to specific glycan epitopes at the monoclonal level is limited, especially in individuals who can control M. tuberculosis infection. We generated human IgG mAbs to AM/LAM from B cells of two asymptomatic individuals exposed to or latently infected with M. tuberculosis. Here, we show that two of these mAbs have high affinity to AM/LAM, are non-competing, and recognize different glycan epitopes distinct from other anti-AM/LAM mAbs reported. Both mAbs recognize virulent M. tuberculosis and nontuberculous mycobacteria with marked differences, can be used for the detection of urinary LAM, and can detect M. tuberculosis and LAM in infected lungs. These mAbs enhance our understanding of the spectrum of antibodies to AM/LAM epitopes in humans and are valuable for tuberculosis diagnostic and research applications.

A Combination of Receptor-Binding Domain and N-Terminal Domain Neutralizing Antibodies Limits the Generation of SARS-CoV-2 Spike Neutralization-Escape Mutants.

Most known SARS-CoV-2 neutralizing antibodies (nAbs), including those approved by the FDA for emergency use, inhibit viral infection by targeting the receptor-binding domain (RBD) of the spike (S) protein. Variants of concern (VOC) carrying mutations in the RBD or other regions of S reduce the effectiveness of many nAbs and vaccines by evading neutralization. Therefore, therapies that are less susceptible to resistance are urgently needed. Here, we characterized the memory B-cell repertoire of COVID-19 convalescent donors and analyzed their RBD and non-RBD nAbs. We found that many of the non-RBD-targeting nAbs were specific to the N-terminal domain (NTD). Using neutralization assays with authentic SARS-CoV-2 and a recombinant vesicular stomatitis virus carrying SARS-CoV-2 S protein (rVSV-SARS2), we defined a panel of potent RBD and NTD nAbs. Next, we used a combination of neutralization-escape rVSV-SARS2 mutants and a yeast display library of RBD mutants to map their epitopes. The most potent RBD nAb competed with hACE2 binding and targeted an epitope that includes residue F490. The most potent NTD nAb epitope included Y145, K150, and W152. As seen with some of the natural VOC, the neutralization potencies of COVID-19 convalescent-phase sera were reduced by 4- to 16-fold against rVSV-SARS2 bearing Y145D, K150E, or W152R spike mutations. Moreover, we found that combining RBD and NTD nAbs did not enhance their neutralization potential. Notably, the same combination of RBD and NTD nAbs limited the development of neutralization-escape mutants in vitro, suggesting such a strategy may have higher efficacy and utility for mitigating the emergence of VOC. IMPORTANCE The U.S. FDA has issued emergency use authorizations (EUAs) for multiple investigational monoclonal antibody (MAb) therapies for the treatment of mild to moderate COVID-19. These MAb therapeutics are solely targeting the receptor-binding domain of the SARS-CoV-2 spike protein. However, the N-terminal domain of the spike protein also carries crucial neutralizing epitopes. Here, we show that key mutations in the N-terminal domain can reduce the neutralizing capacity of convalescent-phase COVID-19 sera. We report that a combination of two neutralizing antibodies targeting the receptor-binding and N-terminal domains may be beneficial to combat the emergence of virus variants.

Near-germline human monoclonal antibodies neutralize and protect against multiple arthritogenic alphaviruses.

Arthritogenic alphaviruses are globally distributed, mosquito-transmitted viruses that cause rheumatological disease in humans and include Chikungunya virus (CHIKV), Mayaro virus (MAYV), and others. Although serological evidence suggests that some antibody-mediated heterologous immunity may be afforded by alphavirus infection, the extent to which broadly neutralizing antibodies that protect against multiple arthritogenic alphaviruses are elicited during natural infection remains unknown. Here, we describe the isolation and characterization of MAYV-reactive alphavirus monoclonal antibodies (mAbs) from a CHIKV-convalescent donor. We characterized 33 human mAbs that cross-reacted with CHIKV and MAYV and engaged multiple epitopes on the E1 and E2 glycoproteins. We identified five mAbs that target distinct regions of the B domain of E2 and potently neutralize multiple alphaviruses with differential breadth of inhibition. These broadly neutralizing mAbs (bNAbs) contain few somatic mutations and inferred germline-revertants retained neutralizing capacity. Two bNAbs, DC2.M16 and DC2.M357, protected against both CHIKV- and MAYV-induced musculoskeletal disease in mice. These findings enhance our understanding of the cross-reactive and cross-protective antibody response to human alphavirus infections.

Pan-protective anti-alphavirus human antibodies target a conserved E1 protein epitope.

Alphaviruses are emerging, mosquito-transmitted pathogens that cause musculoskeletal and neurological disease in humans. Although neutralizing antibodies that inhibit individual alphaviruses have been described, broadly reactive antibodies that protect against both arthritogenic and encephalitic alphaviruses have not been reported. Here, we identify DC2.112 and DC2.315, two pan-protective yet poorly neutralizing human monoclonal antibodies (mAbs) that avidly bind to viral antigen on the surface of cells infected with arthritogenic and encephalitic alphaviruses. These mAbs engage a conserved epitope in domain II of the E1 protein proximal to and within the fusion peptide. Treatment with DC2.112 or DC2.315 protects mice against infection by both arthritogenic (chikungunya and Mayaro) and encephalitic (Venezuelan, Eastern, and Western equine encephalitis) alphaviruses through multiple mechanisms, including inhibition of viral egress and monocyte-dependent Fc effector functions. These findings define a conserved epitope recognized by weakly neutralizing yet protective antibodies that could be targeted for pan-alphavirus immunotherapy and vaccine design.

Single-Dilution COVID-19 Antibody Test with Qualitative and Quantitative Readouts.

The coronavirus disease 2019 (COVID-19) global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to place an immense burden on societies and health care systems. A key component of COVID-19 control efforts is serological testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior SARS-CoV-2 infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this sensitive test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test make it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.IMPORTANCE Serological surveillance has become an important public health tool during the COVID-19 pandemic. Detection of protective antibodies and seroconversion after SARS-CoV-2 infection or vaccination can help guide patient care plans and public health policies. Serology tests can detect antibodies against past infections; consequently, they can help overcome the shortcomings of molecular tests, which can detect only active infections. This is important, especially when considering that many COVID-19 patients are asymptomatic. In this study, we describe an enzyme-linked immunosorbent assay (ELISA)-based qualitative and quantitative serology test developed to measure IgG and IgA antibodies against the SARS-CoV-2 spike glycoprotein. The test can be deployed using commonly available laboratory reagents and equipment and displays high specificity and sensitivity. Furthermore, we demonstrate that IgG titers in patient samples can be estimated from a single measurement, enabling the assay's use in high-throughput clinical environments.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Qualitative Immunoglobulin G Assays: The Value of Numeric Reporting.

CONTEXT.­: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin G (IgG) testing is used for serosurveillance and will be important to evaluate vaccination status. Given the urgency to release coronavirus disease 2019 (COVID-19) serology tests, most manufacturers have developed qualitative tests. OBJECTIVE.­: To evaluate clinical performance of 6 different SARS-CoV-2 IgG assays and their quantitative results to better elucidate the clinical role of serology testing in COVID-19. DESIGN.­: Six SARS-CoV-2 IgG assays were tested using remnant specimens from 190 patients. Sensitivity and specificity were evaluated for each assay with the current manufacturer's cutoff and a lower cutoff. A numeric result analysis and discrepancy analysis were performed. RESULTS.­: Specificity was higher than 93% for all assays, and sensitivity was higher than 80% for all assays (≥7 days post-polymerase chain reaction testing). Inpatients with more severe disease had higher numeric values compared with health care workers with mild or moderate disease. Several discrepant serology results were those just below the manufacturers' cutoff. CONCLUSIONS.­: Severe acute respiratory syndrome coronavirus 2 IgG antibody testing can aid in the diagnosis of COVID-19, especially with negative polymerase chain reaction. Quantitative COVID-19 IgG results are important to better understand the immunologic response and disease course of this novel virus and to assess immunity as part of future vaccination programs.

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.

Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased protein expression. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high-quality S protein (nonaggregated, uniform material with appropriate biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM structures reveals conformation plasticity in the region composed of amino acids 614-642 and 828-854. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome and report no novel binding partners and notably fail to validate the Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural, and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

Treatment of severe COVID-19 with convalescent plasma in Bronx, NYC.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as a treatment for coronavirus disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200 mL of CCP with a spike protein IgG titer ≥ 1:2430 (median 1:47,385) within 72 hours of admission with propensity score-matched controls cared for at a medical center in the Bronx, between April 13 and May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroid use, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared with matched controls, CCP recipients less than 65 years had 4-fold lower risk of mortality and 4-fold lower risk of deterioration in oxygenation or mortality at day 28. For CCP recipients, pretransfusion spike protein IgG, IgM, and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients less than 65 years, but data from controlled trials are needed to validate this finding and establish the effect of aging on CCP efficacy.

Treatment of Severe COVID-19 with Convalescent Plasma in the Bronx, NYC.

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as treatment for Coronavirus Disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200mL of CCP with a Spike protein IgG titer ≥1:2,430 (median 1:47,385) within 72 hours of admission to propensity score-matched controls cared for at a medical center in the Bronx, between April 13 to May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroids, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared to matched controls, CCP recipients <65 years had 4-fold lower mortality and 4-fold lower deterioration in oxygenation or mortality at day 28. For CCP recipients, pre-transfusion Spike protein IgG, IgM and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients <65 years, but data from controlled trials is needed to validate this finding and establish the effect of ageing on CCP efficacy.

Development, clinical translation, and utility of a COVID-19 antibody test with qualitative and quantitative readouts.

The COVID-19 global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to place an immense burden on societies and healthcare systems. A key component of COVID-19 control efforts is serologic testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test makes it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.

A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, define correlates of immune protection, and down-select candidate antivirals. Here, we generate a highly infectious recombinant vesicular stomatitis virus (VSV) bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein and show that this recombinant virus, rVSV-SARS-CoV-2 S, closely resembles SARS-CoV-2 in its entry-related properties. The neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in a high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S, and neutralization of rVSV-SARS-CoV-2 S and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific therapeutics and for mechanistic studies of viral entry and its inhibition.

A replication-competent vesicular stomatitis virus for studies of SARS-CoV-2 spike-mediated cell entry and its inhibition.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis.

Coronavirus disease 2019 ( COVID-19 ) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike ( S ) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F ™ and ExpiCHO-S ™ cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S ™ cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural ( cryo-EM ) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.

Peptide-Based Vaccines: Current Progress and Future Challenges.

Vaccines have had a profound impact on the management and prevention of infectious disease. In addition, the development of vaccines against chronic diseases has attracted considerable interest as an approach to prevent, rather than treat, conditions such as cancer, Alzheimer's disease, and others. Subunit vaccines consist of nongenetic components of the infectious agent or disease-related epitope. In this Review, we discuss peptide-based vaccines and their potential in three therapeutic areas: infectious disease, Alzheimer's disease, and cancer. We discuss factors that contribute to vaccine efficacy and how these parameters may potentially be modulated by design. We examine both clinically tested vaccines as well as nascent approaches and explore current challenges and potential remedies. While peptide vaccines hold substantial promise in the prevention of human disease, many obstacles remain that have hampered their clinical use; thus, continued research efforts to address these challenges are warranted.

Human monoclonal antibodies against chikungunya virus target multiple distinct epitopes in the E1 and E2 glycoproteins.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes persistent arthritis in a subset of human patients. We report the isolation and functional characterization of monoclonal antibodies (mAbs) from two patients infected with CHIKV in the Dominican Republic. Single B cell sorting yielded a panel of 46 human mAbs of diverse germline lineages that targeted epitopes within the E1 or E2 glycoproteins. MAbs that recognized either E1 or E2 proteins exhibited neutralizing activity. Viral escape mutations localized the binding epitopes for two E1 mAbs to sites within domain I or the linker between domains I and III; and for two E2 mAbs between the ß-connector region and the B-domain. Two of the E2-specific mAbs conferred protection in vivo in a stringent lethal challenge mouse model of CHIKV infection, whereas the E1 mAbs did not. These results provide insight into human antibody response to CHIKV and identify candidate mAbs for therapeutic intervention.