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Lassa fever continues to be a major public health burden in West Africa, yet effective therapies or vaccines are lacking. The isolation of protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccine candidates have generally been unsuccessful at doing so, and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron microscopy-based epitope mapping workflow that enables high-resolution structural characterization of polyclonal antibodies to the GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization that involve epitopes of the GPC-A competition cluster. Furthermore, by identifying undescribed immunogenic off-target epitopes, we expose the challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design.
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Broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope glycoprotein (Env) have the capacity to delay viral rebound when administered to people with HIV-1 (PWH) during anti-retroviral therapy (ART) interruption. To further enhance the performance of bNAbs through their Fc effector functions, in particular NK cell-mediated killing of HIV-1 infected cells, we have produced a panel of glyco-engineered (afucosylated) bNAbs with enhanced affinity for Fc gamma receptor IIIa. These afucosylated anti-HIV-1 bNAbs enhance NK cell activation and degranulation compared to fucosylated counterparts even at low antigen density. NK cells from PWH expressing exhaustion markers PD-1 and TIGIT are activated in a similar fashion by afucosylated bNAbs as NK cell from HIV-1 negative individuals. Killing of HIV-1 infected cells is most effective with afucosylated bNAbs 2G12, N6, PGT151 and PGDM1400, whereas afucosylated PGT121 and non-neutralizing antibody A32 only induce minor NK cell-mediated killing. These data indicate that the approach angle and affinity of Abs influence the capacity to induce antibody-dependent cellular cytotoxicity. Thus, afucosylated bNAbs have the capacity to induce NK cell-mediated killing of infected cells, which warrants further investigation of afucosylated bNAb administration in vivo, aiming for reduction of the viral reservoir and ART free durable control.
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Anticorpos Amplamente Neutralizantes , Anticorpos Anti-HIV , Infecções por HIV , HIV-1 , Células Matadoras Naturais , Humanos , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , Anticorpos Anti-HIV/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Neutralizantes/imunologia , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/imunologia , FucoseRESUMO
Gold nanoparticles (AuNPs) decorated with antigens have recently emerged as promising tools for vaccine development due to their innate ability to provide stability to antigens and modulate immune responses. In this study, we have engineered deactivated virus-like particles (VLPs) by precisely functionalizing gold cores with coronas comprising the full SARS-CoV-2 spike protein (S). Using BALB/c mice as a model, we investigated the immunogenicity of these S-AuNPs-VLPs. Our results demonstrate that S-AuNPs-VLPs consistently enhanced antigen-specific antibody responses compared to the S protein free in solution. This enhancement included higher binding antibody titers, higher neutralizing capacity of antibodies, and stronger T-cell responses. Compared to the mRNA COVID-19 vaccine, where the S protein is synthesized in situ, S-AuNPs-VLPs induced comparable binding and neutralizing antibody responses, but substantially superior T-cell responses. In conclusion, our study highlights the potential of conjugated AuNPs as an effective antigen-delivery system for protein-based vaccines targeting a broad spectrum of infectious diseases and other emergent viruses.
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BACKGROUND: The immunological determinants of delayed viral clearance and intra-host viral evolution that drive the development of new pathogenic virus strains in immunocompromised individuals are unknown. Therefore, we longitudinally studied SARS-CoV-2-specific immune responses in relation to viral-clearance and evolution in immunocompromised individuals. METHODS: Among Omicron-infected immunocompromised individuals, we determined SARS-CoV-2-specific T- and B-cell responses, anti-spike IgG(3) titers, neutralization titers, and monoclonal antibody (mAb)-resistance-associated mutations. The 28-day post-enrollment nasopharyngeal specimen defined early (RT-PCR negative ≤28 days) or late (RT-PCR- positive >28 days) viral-clearance. RESULTS: Of 30 patients included (median age 61.9 years [IQR 47.4-72.3], 50% females), 20 (66.7%) received mAb-therapy. Thirteen (43.3%) demonstrated early and 17 (56.7%) late viral-clearance. Early viral-clearance patients and patients without resistance-associated mutations had significantly higher baseline IFN-γ release and early viral-clearance patients had a higher frequency of SARS-CoV-2-specific B-cells at baseline. In non-mAb-treated patients, day 7 IgG and neutralization titers were significantly higher in those with early versus late viral-clearance. CONCLUSION: An early robust adaptive immune response is vital for efficient viral-clearance and associated with less emergence of mAb-resistance-associated mutations in Omicron-infected immunocompromised patients. This emphasizes the importance of early SARS-CoV-2-specific T- and B-cell responses and thereby provides a rationale for development of novel therapeutic approaches.
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Recombinant native-like HIV-1 envelope glycoprotein (Env) trimers are used in candidate vaccines aimed at inducing broadly neutralizing antibodies. While state-of-the-art SOSIP or single-chain Env designs can be expressed as native-like trimers, undesired monomers, dimers and malformed trimers that elicit non-neutralizing antibodies are also formed, implying that these designs could benefit from further modifications for gene-based vaccination approaches. Here, we describe the triple tandem trimer (TTT) design, in which three Env protomers are genetically linked in a single open reading frame and express as native-like trimers. Viral vectored Env TTT induced similar neutralization titers but with a higher proportion of trimer-specific responses. The TTT design was also applied to generate influenza hemagglutinin (HA) trimers without the need for trimerization domains. Additionally, we used TTT to generate well-folded chimeric Env and HA trimers that harbor protomers from three different strains. In summary, the TTT design is a useful platform for the design of HIV-1 Env and influenza HA immunogens for a multitude of vaccination strategies.
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One of the strategies towards an effective HIV-1 vaccine is to elicit broadly neutralizing antibody responses that target the high HIV-1 Env diversity. Here, we present an HIV-1 vaccine candidate that consists of cobalt porphyrin-phospholipid (CoPoP) liposomes decorated with repaired and stabilized clade C HIV-1 Env trimers in a prefusion conformation. These particles exhibit high HIV-1 Env trimer decoration, serum stability and bind broadly neutralizing antibodies. Three sequential immunizations of female rabbits with CoPoP liposomes displaying a different clade C HIV-1 gp140 trimer at each dosing generate high HIV-1 Env-specific antibody responses. Additionally, serum neutralization is detectable against 18 of 20 multiclade tier 2 HIV-1 strains. Furthermore, the peak antibody titers induced by CoPoP liposomes can be recalled by subsequent heterologous immunization with Ad26-encoded membrane-bound stabilized Env antigens. Hence, a CoPoP liposome-based HIV-1 vaccine that can generate cross-clade neutralizing antibody immunity could potentially be a component of an efficacious HIV-1 vaccine.
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Vacinas contra a AIDS , HIV-1 , Produtos do Gene env do Vírus da Imunodeficiência Humana , Animais , Feminino , Coelhos , Anticorpos Neutralizantes , Anticorpos Anti-HIV , Infecções por HIV , Imunização , Lipossomos , FosfolipídeosRESUMO
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has remained a medical threat due to the evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants. A stabilized spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants, with COVA309-35 being the most potent against the autologous virus, as well as Omicron BA.1 and BA.2, and COVA309-22 having binding and neutralization activity against Omicron BA.4/5, BQ.1.1, and XBB.1. When combining the COVA309 mAbs as cocktails or bispecific antibodies, the breadth and potency were improved. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.
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OBJECTIVE: People with HIV rarely control viral replication after cessation of antiretroviral therapy (ART). We present a person with HIV with extraordinary posttreatment control (PTC) for over 23âyears after temporary ART during acute HIV infection (AHI) leading to a new insight in factors contributing to PTC. DESIGN/METHODS: Viral reservoir was determined by HIV qPCR, Intact Proviral DNA Assay, and quantitative viral outgrowth assay. Viral replication kinetics were determined in autologous and donor PBMC. IgG levels directed against HIV envelope and neutralizing antibodies were measured. Immune phenotyping of T cells and HIV-specific T-cell responses were analyzed by flow cytometry. RESULTS: The case presented with AHI and a plasma viral load of 2.7 million copies/ml. ART was initiated 2âweeks after diagnosis and interrupted after 26âmonths. Replicating virus was isolated shortly after start ART. At 18âyears after treatment interruption, HIV-DNA in CD4 + T cells and low levels of HIV-RNA in plasma (<5âcopies/ml) were detectable. Stable HIV envelope glycoprotein-directed IgG was present during follow-up, but lacked neutralizing activity. Strong antiviral CD8 + T-cell responses, in particular targeting HIV-gag, were detected during 25âyears follow-up. Moreover, we found a P255A mutation in an HLA-B∗44â:â02 restricted gag-epitope, which was associated with decreased replication. CONCLUSION: We describe an exceptional case of PTC, which is likely associated with sustained potent gag-specific CD8 + T-cell responses in combination with a replication attenuating escape mutation in gag. Understanding the initiation and preservation of the HIV-specific T-cell responses could guide the development of strategies to induce HIV control.
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Infecções por HIV , Humanos , Leucócitos Mononucleares , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , DNA , Imunoglobulina G , Carga ViralRESUMO
Background: Patients with haematological malignancies have impaired antibody responses to SARS-CoV-2 vaccination. We aimed to investigate whether a fourth mRNA COVID-19 vaccination improved antibody quantity and quality. Methods: In this cohort study, conducted at 5 sites in the Netherlands, we compared antibody concentrations 28 days after 4 mRNA vaccinations (3-dose primary series plus 1 booster vaccination) in SARS-CoV-2 naive, immunocompromised patients with haematological malignancies to those obtained by age-matched, healthy individuals who had received the standard primary 2-dose mRNA vaccination schedule followed by a first booster mRNA vaccination. Prior to and 4 weeks after each vaccination, peripheral blood samples and data on demographic parameters and medical history were collected. Concentrations of antibodies that bind spike 1 (S1) and nucleocapsid (N) protein of SARS-CoV-2 were quantified in binding antibody units (BAU) per mL according to the WHO International Standard for COVID-19 serological tests. Seroconversion was defined as an S1 IgG concentration >10 BAU/mL and a previous SARS-CoV-2 infection as N IgG >14.3 BAU/mL. Antibody neutralising activity was tested using lentiviral-based pseudoviruses expressing spike protein of SARS-CoV-2 wild-type (D614G), Omicron BA.1, and Omicron BA.4/5 variants. This study is registered with EudraCT, number 2021-001072-41. Findings: Between March 24, 2021 and May 4, 2021, 723 patients with haematological diseases were enrolled, of which 414 fulfilled the inclusion criteria for the current analysis. Although S1 IgG concentrations in patients significantly improved after the fourth dose, they remained significantly lower compared to those obtained by 58 age-matched healthy individuals after their first booster (third) vaccination. The rise in neutralising antibody concentration was most prominent in patients with a recovering B cell compartment, although potent responses were also observed in patients with persistent immunodeficiencies. 19% of patients never seroconverted, despite 4 vaccinations. Patients who received their first 2 vaccinations when they were B cell depleted and the third and fourth vaccination during B cell recovery demonstrated similar antibody induction dynamics as patients with normal B cell numbers during the first 2 vaccinations. However, the neutralising capacity of these antibodies was significantly better than that of patients with normal B cell numbers after two vaccinations. Interpretation: A fourth mRNA COVID-19 vaccination improved S1 IgG concentrations in the majority of patients with a haematological malignancy. Vaccination during B cell depletion may pave the way for better quality of antibody responses after B cell reconstitution. Funding: The Netherlands Organisation for Health Research and Development and Amsterdam UMC.
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Lassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences among phylogenetically distinct LASV lineages. Despite the sequence diversity of the GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VII, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of the GPC in complex with GP1-A-specific antibodies suggest their neutralization mechanisms. Finally, we present the isolation and characterization of a trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines.
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Febre Lassa , Vírus Lassa , Humanos , Anticorpos Neutralizantes , Febre Lassa/prevenção & controle , Glicoproteínas , Antígenos ViraisRESUMO
SARS-CoV-2 variants evade current monoclonal antibody therapies. Bispecific antibodies (bsAbs) combine the specificities of two distinct antibodies taking advantage of the avidity and synergy provided by targeting different epitopes. Here we used controlled Fab-arm exchange to produce bsAbs that neutralize SARS-CoV and SARS-CoV-2 variants, including Omicron and its subvariants, by combining potent SARS-CoV-2-specific neutralizing antibodies with broader antibodies that also neutralize SARS-CoV. We demonstrated that the parental antibodies rely on avidity for neutralization using bsAbs containing one irrelevant Fab arm. Using mass photometry to measure the formation of antibody:spike complexes, we determined that bsAbs increase binding stoichiometry compared to corresponding cocktails, without a loss of binding affinity. The heterogeneous binding pattern of bsAbs to spike, observed by negative-stain electron microscopy and mass photometry provided evidence for both intra- and inter-spike crosslinking. This study highlights the utility of cross-neutralizing antibodies for designing bivalent agents to combat circulating and future SARS-like coronaviruses.
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Targeting germline (gl-) precursors of broadly neutralizing antibodies (bNAbs) is acknowledged as an important strategy for HIV-1 vaccines. The VRC01-class of bNAbs is attractive because of its distinct genetic signature. However, VRC01-class bNAbs often require extensive somatic hypermutation, including rare insertions and deletions. We describe a BG505 SOSIP trimer, termed GT1.2, to optimize binding to gl-CH31, the unmutated common precursor of the CH30-34 bNAb lineage that acquired a large CDRH1 insertion. The GT1.2 trimer activates gl-CH31 naive B cells in knock-in mice, and B cell responses could be matured by selected boosting immunogens to generate cross-reactive Ab responses. Next-generation B cell sequencing reveals selection for VRC01-class mutations, including insertions in CDRH1 and FWR3 at positions identical to VRC01-class bNAbs, as well as CDRL1 deletions and/or glycine substitutions to accommodate the N276 glycan. These results provide proof of concept for vaccine-induced affinity maturation of B cell lineages that require rare insertions and deletions.
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Soropositividade para HIV , HIV-1 , Camundongos , Animais , Anticorpos Amplamente Neutralizantes , Anticorpos Neutralizantes , HIV-1/genética , Anticorpos Anti-HIV , VacinaçãoRESUMO
Lassa fever continues to be a major public health burden in endemic countries in West Africa, yet effective therapies or vaccines are lacking. The isolation of potent and protective neutralizing antibodies against the Lassa virus glycoprotein complex (GPC) justifies the development of vaccines that can elicit strong neutralizing antibody responses. However, Lassa vaccines candidates have generally been unsuccessful in doing so and the associated antibody responses to these vaccines remain poorly characterized. Here, we establish an electron-microscopy based epitope mapping pipeline that enables high-resolution structural characterization of polyclonal antibodies to GPC. By applying this method to rabbits vaccinated with a recombinant GPC vaccine and a GPC-derived virus-like particle, we reveal determinants of neutralization which involve epitopes of the GPC-C, GPC-A, and GP1-A competition clusters. Furthermore, by identifying previously undescribed immunogenic off-target epitopes, we expose challenges that recombinant GPC vaccines face. By enabling detailed polyclonal antibody characterization, our work ushers in a next generation of more rational Lassa vaccine design.
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Broadly neutralizing antibodies (bNAbs) have remarkable breadth and potency against most HIV-1 subtypes and are able to prevent HIV-1 infection in animal models. However, bNAbs are extremely difficult to induce by vaccination. Defining the developmental pathways towards neutralization breadth can assist in the design of strategies to elicit protective bNAb responses by vaccination. Here, HIV-1 envelope glycoproteins (Env)-specific IgG+ B cells were isolated at various time points post infection from an HIV-1 infected elite neutralizer to obtain monoclonal antibodies (mAbs). Multiple antibody lineages were isolated targeting distinct epitopes on Env, including the gp120-gp41 interface, CD4-binding site, silent face and V3 region. The mAbs each neutralized a diverse set of HIV-1 strains from different clades indicating that the patient's remarkable serum breadth and potency might have been the result of a polyclonal mixture rather than a single bNAb lineage. High-resolution cryo-electron microscopy structures of the neutralizing mAbs (NAbs) in complex with an Env trimer generated from the same individual revealed that the NAbs used multiple strategies to neutralize the virus; blocking the receptor binding site, binding to HIV-1 Env N-linked glycans, and disassembly of the trimer. These results show that diverse NAbs can complement each other to achieve a broad and potent neutralizing serum response in HIV-1 infected individuals. Hence, the induction of combinations of moderately broad NAbs might be a viable vaccine strategy to protect against a wide range of circulating HIV-1 viruses.
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Soropositividade para HIV , HIV-1 , Animais , Anticorpos Amplamente Neutralizantes , Microscopia Crioeletrônica , Anticorpos Monoclonais , Proteína gp120 do Envelope de HIVRESUMO
The Lassa virus is endemic in parts of West Africa, and it causes hemorrhagic fever with high mortality. The development of a recombinant protein vaccine has been hampered by the instability of soluble Lassa virus glycoprotein complex (GPC) trimers, which disassemble into monomeric subunits after expression. Here, we use two-component protein nanoparticles consisting of trimeric and pentameric subunits to stabilize GPC in a trimeric conformation. These GPC nanoparticles present twenty prefusion GPC trimers on the surface of an icosahedral particle. Cryo-EM studies of GPC nanoparticles demonstrated a well-ordered structure and yielded a high-resolution structure of an unliganded GPC. These nanoparticles induced potent humoral immune responses in rabbits and protective immunity against the lethal Lassa virus challenge in guinea pigs. Additionally, we isolated a neutralizing antibody that mapped to the putative receptor-binding site, revealing a previously undefined site of vulnerability. Collectively, these findings offer potential approaches to vaccine and therapeutic design for the Lassa virus.
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Febre Lassa , Nanopartículas , Cobaias , Coelhos , Animais , Vírus Lassa/química , Anticorpos Neutralizantes , Febre Lassa/prevenção & controle , Glicoproteínas , Vacinas SintéticasRESUMO
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses continuous challenges in combating the virus. Here, we describe vaccination strategies to broaden SARS-CoV-2 and sarbecovirus immunity by combining spike proteins based on different viruses or viral strains displayed on two-component protein nanoparticles. First, we combined spike proteins based on ancestral and Beta SARS-CoV-2 strains to broaden SARS-CoV-2 immune responses. Inclusion of Beta spike improved neutralizing antibody responses against SARS-CoV-2 Beta, Gamma, and Omicron BA.1 and BA.4/5. A third vaccination with ancestral SARS-CoV-2 spike also improved cross-neutralizing antibody responses against SARS-CoV-2 variants, in particular against the Omicron sublineages. Second, we combined SARS-CoV and SARS-CoV-2 spike proteins to broaden sarbecovirus immune responses. Adding SARS-CoV spike to a SARS-CoV-2 spike vaccine improved neutralizing responses against SARS-CoV and SARS-like bat sarbecoviruses SHC014 and WIV1. These results should inform the development of broadly active SARS-CoV-2 and pan-sarbecovirus vaccines and highlight the versatility of two-component nanoparticles for displaying diverse antigens.
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The worldwide pandemic caused by SARS-CoV-2 has remained a human medical threat due to the continued evolution of multiple variants that acquire resistance to vaccines and prior infection. Therefore, it is imperative to discover monoclonal antibodies (mAbs) that neutralize a broad range of SARS-CoV-2 variants for therapeutic and prophylactic use. A stabilized autologous SARS-CoV-2 spike glycoprotein was used to enrich antigen-specific B cells from an individual with a primary Gamma variant infection. Five mAbs selected from those B cells showed considerable neutralizing potency against multiple variants of concern, with COVA309-35 being the most potent against the autologous virus, as well as against Omicron BA.1 and BA.2. When combining the COVA309 mAbs as cocktails or bispecific antibody formats, the breadth and potency was significantly improved against all tested variants. In addition, the mechanism of cross-neutralization of the COVA309 mAbs was elucidated by structural analysis. Altogether these data indicate that a Gamma-infected individual can develop broadly neutralizing antibodies.
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Antibodies against seasonal human coronaviruses (HCoVs) are known to cross-react with SARS-CoV-2, but data on cross-protective effects of prior HCoV infections are conflicting. In a prospective cohort of healthcare workers (HCWs), we studied the association between seasonal HCoV (OC43, HKU1, 229E and NL63) nucleocapsid protein IgG and SARS-CoV-2 infection during the first pandemic wave in the Netherlands (March 2020 - June 2020), by 4-weekly serum sampling. HCW with HCoV-OC43 antibody levels in the highest quartile, were less likely to become SARS-CoV-2 seropositive when compared with those with lower levels (6/32, 18.8%, versus 42/97, 43.3%, respectively: p = 0.019; HR 0.37, 95% CI 0.16-0.88). We found no significant association with HCoV-OC43 spike protein IgG, or with antibodies against other HCoVs. Our results indicate that the high levels of HCoV-OC43-nucleocapsid antibodies, as an indicator of a recent infection, are associated with protection against SARS-CoV-2 infection; this supports and informs efforts to develop pancoronavirus vaccines.
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Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigate the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We show that â¼82% of SARS-CoV-2 S-reactive B cells harbor a naive phenotype, which represents an unusually high fraction of total human naive B cells (â¼0.1%). Approximately 10% of these naive S-reactive B cells share an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.
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COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , Epitopos , Humanos , Isotipos de Imunoglobulinas , Receptores de Antígenos de Linfócitos B , Glicoproteína da Espícula de CoronavírusRESUMO
Importance: It has become common practice to offer immunocompromised patients with hematologic cancers a third COVID-19 vaccination dose, but data substantiating this are scarce. Objective: To assess whether a third mRNA-1273 vaccination is associated with increased neutralizing antibody concentrations in immunocompromised patients with hematologic cancers comparable to levels obtained in healthy individuals after the standard 2-dose mRNA-1273 vaccination schedule. Design, Setting, and Participants: This prospective observational cohort study was conducted at 4 university hospitals in the Netherlands and included 584 evaluable patients spanning the spectrum of hematologic cancers and 44 randomly selected age-matched adults without malignant or immunodeficient comorbidities. Exposures: One additional mRNA-1273 vaccination 5 months after completion of the standard 2-dose mRNA-1273 vaccination schedule. Main Outcomes and Measures: Serum immunoglobulin G (IgG) antibodies to spike subunit 1 (S1) antigens prior to and 4 weeks after a third mRNA-1273 vaccination, and antibody neutralization capacity of wild-type, Delta, and Omicron variants in a subgroup of patients. Results: In this cohort of 584 immunocompromised patients with hematologic cancers (mean [SD] age, 60 [11.2] years; 216 [37.0%] women), a third mRNA-1273 vaccination was associated with median S1-IgG concentrations comparable to concentrations obtained by healthy individuals after the 2-dose mRNA-1273 schedule. The rise in S1-IgG concentration after the third vaccination was most pronounced in patients with a recovering immune system, but potent responses were also observed in patients with persistent immunodeficiencies. Specifically, patients with myeloid cancers or multiple myeloma and recipients of autologous or allogeneic hematopoietic cell transplantation (HCT) reached median S1-IgG concentrations similar to those obtained by healthy individuals after a 2-dose schedule. Patients receiving or shortly after completing anti-CD20 therapy, CD19-directed chimeric antigen receptor T-cell therapy recipients, and patients with chronic lymphocytic leukemia receiving ibrutinib were less responsive or unresponsive to the third vaccination. In the 27 patients who received cell therapy between the second and third vaccination, S1 antibodies were preserved, but a third mRNA-1273 vaccination was not associated with significantly enhanced S1-IgG concentrations except for patients with multiple myeloma receiving autologous HCT. A third vaccination was associated with significantly improved neutralization capacity per antibody. Conclusions and Relevance: Results of this cohort study support that the primary schedule for immunocompromised patients with hematologic cancers should be supplemented with a delayed third vaccination. Patients with B-cell lymphoma and allogeneic HCT recipients need to be revaccinated after treatment or transplantation. Trial Registration: EudraCT Identifier: 2021-001072-41.