Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviral-vectored COVID-19 vaccines. VITT had been linked to production of autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact mass measurements indicate that a significant fraction of these antibodies represent a limited number of clones. MS analysis of large antibody fragments (the light chain and the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire and reveals the presence of a mature complex biantennary N-glycan within the Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS was used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to the IgG2 subclass and verifies that the light chain belongs to the λ-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site is the result of a single mutation within the germline sequence. Peptide mapping also provides information on lower-abundance (polyclonal) components of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1-IgG4) and both types of the light chain (λ and κ). This case study demonstrates the power of combining the intact, middle-down, and bottom-up MS approaches for meaningful characterization of ultralow quantities of pathogenic antibodies extracted directly from patients' blood.

2023 ISTH update of the 2022 ISTH guidelines for antithrombotic treatment in COVID-19.

Based on emerging evidence from the COVID-19 pandemic, the International Society on Thrombosis and Haemostasis (ISTH) guidelines for antithrombotic treatment in COVID-19 were published in 2022. Since then, at least 16 new randomized controlled trials have contributed additional evidence, which necessitated a modification of most of the previous recommendations. We used again the American College of Cardiology Foundation/American Heart Association methodology for assessment of level of evidence (LOE) and class of recommendation (COR). Five recommendations had the LOE upgraded to A and 2 new recommendations on antithrombotic treatment for patients with COVID-19 were added. Furthermore, a section was added to answer questions about COVID-19 vaccination and vaccine-induced immune thrombotic thrombocytopenia (VITT), for which studies have provided some evidence. We only included recommendations with LOE A or B. Panelists agreed on 19 recommendations, 4 for nonhospitalized, 5 for noncritically ill hospitalized, 3 for critically ill hospitalized, and 2 for postdischarge patients, as well as 5 for vaccination and VITT. A strong recommendation (COR 1) was given for (a) use of prophylactic dose of low-molecular-weight heparin or unfractionated heparin in noncritically ill patients hospitalized for COVID-19, (b) for select patients in this group, use of therapeutic-dose low-molecular-weight heparin/unfractionated heparin in preference to prophylactic dose, and (c) for use of antiplatelet factor 4 enzyme immunoassays for diagnosing VITT. A strong recommendation was given against (COR 3) the addition of an antiplatelet agent in hospitalized, noncritically ill patients. These international guidelines provide recommendations for countries with diverse healthcare resources and COVID-19 vaccine availability.

Bleeding self-assessments by patients with immune thrombocytopenia (ITP): An agreement study.

We designed anagreement study to compare the results of bleeding assessments done in tandem by ITP patients and trained research staff. We used a modified version of the ITP Bleeding Scale, which captured the patients' worst bleeding event at any of nine anatomical sites since the time of the last assessment. Interrater agreement was determined using the 2-way kappa for the assessment of severe vs. non-severe bleeds. We analyzed 108 consecutive patients with ITP from the McMaster ITP Registry who had duplicate bleeding assessments. Two-way agreement was excellent for gynecological (k = 0.86, 95% CI 0.71-1.02), gastrointestinal (k = 1), genitourinary (k = 1), pulmonary (k = 1) and intracranial (k = 1) bleeds; good for skin (k = 0.68, 95% CI, 0.54-0.82), oral (k = 0.76, 95% CI, 0.53-0.98) and ocular (k = 0.66, 95% CI, 0.04-1-28) bleeds; and moderate for epistaxis (k = 0.58, 95% CI, 0.21-0.95). Bleeding self-assessments by ITP patients were similar to trained research staff, but disagreements in severity grades were more frequent with skin bleeds, oral bleeds and epistaxis. Bleeding self-assessments could simplify bleeding assessments in clinical trials.

Molecular architecture and platelet-activating properties of small immune complexes assembled on heparin and platelet factor 4.

Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin leading to a reduction in circulating platelets with an increased risk of thrombosis. It is precipitated by polymerized immune complexes consisting of pathogenic antibodies that recognize a small chemokine platelet factor 4 (PF4) bound to heparin. Characterization of these immune complexes is extremely challenging due to the enormous structural heterogeneity of such macromolecular assemblies and their constituents. Native mass spectrometry demonstrates that up to three PF4 tetramers can be assembled on a heparin chain, consistent with the molecular modeling studies showing facile polyanion wrapping along the polycationic belt on the PF4 surface. Although these assemblies can accommodate a maximum of only two antibodies, the resulting immune complexes are capable of platelet activation despite their modest size. Taken together, these studies provide further insight into molecular mechanisms of HIT and other immune disorders where anti-PF4 antibodies play a central role.

Vaccine-Induced Immune Thrombotic Thrombocytopenia: Clinicopathologic Features and New Perspectives on Anti-PF4 Antibody-Mediated Disorders.

INTRODUCTION: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet severe adverse complication first identified during the global vaccination effort against SARS-CoV-2 infection, predominantly observed following administration of the ChAdOx1-S (Oxford-AstraZeneca) and Ad26.CoV2.S (Johnson & Johnson/Janssen) adenoviral vector-based vaccines. Unlike other anti-platelet factor 4 (PF4) antibody-mediated disorders, such as heparin-induced thrombocytopenia (HIT), VITT arises with the development of platelet-activating anti-PF4 antibodies 4-42 days post-vaccination, typically featuring thrombocytopenia and thrombosis at unusual sites. AIM: To explore the unique properties, pathogenic mechanisms, and long-term persistence of VITT antibodies in patients, in comparison with other anti-PF4 antibody-mediated disorders. DISCUSSION: This review highlights the complexity of VITT as it differs in antibody behavior and clinical presentation from other anti-PF4-mediated disorders, including the high incidence rate of cerebral venous sinus thrombosis (CVST) and the persistence of anti-PF4 antibodies, necessitating a re-evaluation of long-term patient care strategies. The nature of VITT antibodies and the underlying mechanisms triggering their production remain largely unknown. CONCLUSION: The rise in awareness and subsequent prompt recognition of VITT is paramount in reducing mortality. As vaccination campaigns continue, understanding the role of adenoviral vector-based vaccines in VITT antibody production is crucial, not only for its immediate clinical implications, but also for developing safer vaccines in the future.

Hematology in the post-COVID era: spotlight on vaccine-induced immune thrombotic thrombocytopenia and a conceptual framework (the 4P's) for anti-PF4 diseases.

INTRODUCTION: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a life-threatening prothrombotic disorder first identified following the introduction of adenoviral vector vaccines for COVID-19. The condition is characterized by anti-PF4 antibodies and clinically presents with thrombocytopenia and thrombosis often in unusual anatomical sites. AREAS COVERED: In this review, we discuss the clinical presentation, diagnostic testing, and treatment of VITT. We also review VITT-like syndromes that have been described in patients without previous vaccination. We propose a conceptual framework for the mechanism of anti-PF4 diseases that includes sufficiently high levels of PF4, the presence of a Polyanion that can form immune complexes with PF4, a Pro-inflammatory milieu, and an immunological Predisposition - the 4Ps. EXPERT OPINION: Significant progress has been made in understanding the characteristics of the VITT antibody and in testing methods that can confirm that diagnosis. Future work should be directed at understanding long-term outcomes, mechanisms of thrombosis, and individual risk factors for this rare but dangerous immune-thrombotic disease.

Targeted ADAMTS-13 replacement therapy for thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic disorder associated with a severe deficiency of ADAMTS-13-the protease that cleaves von Willebrand factor. Plasma therapy is the current standard of care for managing acute episodes of TTP, which involves removing patient plasma and replacing it with donor plasma to raise the level of ADAMTS-13 activity. Recently, therapies aimed at replacing ADAMTS-13 have been investigated as possible substitutes or add-ons to plasma therapy for congenital and immune-mediated TTP. Enzyme replacement therapy provides recombinant ADAMTS-13 via intravenous (i.v.) infusion to restore enzyme activity. Recombinant ADAMTS-13-loaded platelets localize to the site of thrombus formation in a more concentrated manner than enzyme replacement or plasma therapy. ADAMTS-13-encoding messenger RNA aims to induce a steady supply of secreted protein and gene therapy is a potentially curative strategy. Overall, targeted ADAMTS-13 replacement therapies may provide better outcomes than plasma therapy by achieving higher levels of ADAMTS-13 activity and a more sustained response with fewer adverse events. Herein, we describe targeted ADAMTS-13 replacement therapies for the treatment of TTP and discuss the advantages and limitations of each approach.

Immunomodulatory drugs have divergent effects on humoral and cellular immune responses to SARS-CoV-2 vaccination in people living with rheumatoid arthritis.

Understanding the efficacy of SARS-CoV-2 vaccination in people on immunosuppressive drugs, including those with rheumatoid arthritis (RA), is critical for their protection. Vaccine induced protection requires antibodies, CD4+ T cells, and CD8+ T cells, but it is unclear if these are equally affected by immunomodulatory drugs. Here, we determined how humoral and cellular SARS-CoV-2 vaccination responses differed between people with RA and controls, and which drug classes impacted these responses. Blood was collected from participants with RA on immunomodulatory drugs and controls after their second, third, and fourth SARS-CoV-2 vaccinations. Receptor binding domain (RBD)-specific antibodies were quantified by ELISA. Spike-specific memory T cells were quantitated using flow cytometry. Linear mixed models assessed the impact of age, sex, and immunomodulatory drug classes on SARS-CoV-2 vaccination responses. Compared to non-RA controls (n = 35), participants with RA on immunomodulatory drugs (n = 62) had lower anti-RBD IgG and spike-specific CD4+ T cell levels, but no deficits in spike-specific CD8+ T cells, following SARS-CoV-2 vaccination. Use of costimulation inhibitors was associated with lower humoral responses. JAK inhibitors were associated with fewer spike-specific CD4+ T cells. Participants with RA on immunomodulatory drugs mounted weaker responses to SARS-CoV-2 vaccination, with different drug classes impacting the cellular and humoral compartments.

Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia.

The massive COVID-19 vaccine roll-out campaign illuminated a range of rare side effects, the most dangerous of which─vaccine-induced immune thrombotic thrombocytopenia (VITT)─is caused by adenoviral (Ad)-vectored vaccines. VITT occurrence had been linked to the production of pathogenic antibodies that recognize an endogenous chemokine, platelet factor 4 (PF4). Mass spectrometry (MS)-based evaluation of the ensemble of anti-PF4 antibodies obtained from a VITT patient's blood indicates that the major component is a monoclonal antibody. Structural characterization of this antibody reveals several unusual characteristics, such as the presence of an N-glycan in the Fab segment and high density of acidic amino acid residues in the complementarity-determining regions. A recombinant version of this antibody (RVT1) was generated by transient expression in mammalian cells based on the newly determined sequence. It captures the key properties of VITT antibodies such as their ability to activate platelets in a PF4 concentration-dependent fashion. Homology modeling of the Fab segment reveals a well-defined polyanionic paratope, and the docking studies indicate that the polycationic segment of PF4 readily accommodates two Fab segments, cross-linking the antibodies to yield polymerized immune complexes. Their existence was verified with native MS by detecting assemblies as large as (RVT1)3(PF4)2, pointing out at FcγRIIa-mediated platelet activation as the molecular mechanism underlying VITT clinical manifestations. In addition to the high PF4 affinity, RVT1 readily binds other polycationic targets, indicating a polyreactive nature of this antibody. This surprising promiscuity not only sheds light on VITT etiology but also opens up a range of opportunities to manage this pathology.

Early humoral and cellular responses after bivalent SARS-CoV-2 mRNA-1273.214 vaccination in long-term care and retirement home residents in Ontario, Canada: An observational cohort study.

Immunogenicity of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) bivalent mRNA-1273.214 vaccine (Original/Omicron B.1.1.529 [BA.1]) is underreported in vulnerable older adults in congregate care settings. In residents of 26 long-term care and retirement homes in Ontario, Canada, humoral (i.e., serum anti-spike and anti-receptor binding domain [anti-RBD]) IgG and IgA antibodies and live SARS-CoV-2 neutralization) and cellular (i.e., CD4+ and CD8+ activation-induced marker spike-specific T cell memory) responses were assessed 7-120 days postvaccination with four monovalent mRNA vaccines (n = 494) or subsequent bivalent mRNA-1273.214 vaccination (fifth vaccine) (n = 557). Within 4 months, anti-spike and anti-RBD antibody levels were similar after monovalent and bivalent vaccination in infection-naïve individuals. Hybrid immunity (i.e., vaccination and natural infection) generally increased humoral responses. After bivalent vaccination, compared to monovalent vaccination, residents with hybrid immunity had elevated anti-spike and anti-RBD IgG and IgA antibodies. Omicron BA.1 antibody-mediated neutralization, and CD8+ T cell memory responses to the Omicron BA.1 spike protein, were also higher after bivalent vaccination. Humoral and cellular responses were, therefore, noninferior within 4 months of bivalent mRNA-1273.214 vaccination compared to monovalent mRNA vaccination. Waning of humoral but not cellular immunity was particularly evident in individuals without hybrid immunity. Continued monitoring of vaccine-associated and hybrid immunity against emerging Omicron variants of concern is necessary to assess longevity of protection.

Early Omicron infection is associated with increased reinfection risk in older adults in long-term care and retirement facilities.

Background: Older adults are at increased risk of SARS-CoV-2 Omicron infection and severe disease, especially those in congregate living settings, despite high SARS-CoV-2 vaccine coverage. It is unclear whether hybrid immunity (combined vaccination and infection) after one Omicron infection provides increased protection against subsequent Omicron reinfection in older adults. Methods: Incidence of SARS-CoV-2 Omicron infection was examined in 750 vaccinated residents of long-term care and retirement homes in the observational cohort COVID in Long-Term Care Study in Ontario, Canada, within a 75-day period (July to September 2022). Risk of infection was assessed by Cox proportional hazards regression. Serum anti-spike and anti-RBD SARS-CoV-2 IgG and IgA antibodies, microneutralization titres, and spike-specific T cell memory responses, were examined in a subset of 318 residents within the preceding three months. Findings: 133 of 750 participants (17.7%) had a PCR-confirmed Omicron infection during the observation period. Increased infection risk was associated with prior Omicron infection (at 9-29 days: 47.67 [23.73-95.76]), and this was not attributed to days since fourth vaccination (1.00 [1.00-1.01]) or residence outbreaks (>6 compared to ≤6: 0.95 [0.37-2.41]). Instead, reinfected participants had lower serum neutralizing antibodies to ancestral and Omicron BA.1 SARS-CoV-2, and lower anti-RBD IgG and IgA antibodies, after their initial Omicron infection. Interpretation: Counterintuitively, SARS-CoV-2 Omicron infection was associated with increased risk of Omicron reinfection in residents of long-term care and retirement homes. Less robust humoral hybrid immune responses in older adults may contribute to risk of Omicron reinfection. Funding: COVID-19 Immunity Task Force of the Public Health Agency of Canada.

Structural characterization of a pathogenic antibody underlying vaccine-induced immune thrombotic thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but extremely dangerous side effect that has been reported for several adenoviral (Ad)-vectored COVID-19 vaccines. VITT pathology had been linked to production of antibodies that recognize platelet factor 4 (PF4), an endogenous chemokine. In this work we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact-mass MS measurements indicate that a significant fraction of this ensemble is comprised of antibodies representing a limited number of clones. MS analysis of large antibody fragments (the light chain, as well as the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire, and reveals the presence of a fully mature complex biantennary N-glycan within its Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS analysis were used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to IgG2 subclass and verify that the light chain belongs to the λ-type. Incorporation of enzymatic de- N -glycosylation into the peptide mapping routine allows the N -glycan in the Fab region of the antibody to be localized to the framework 3 region of the V H domain. This novel N -glycosylation site (absent in the germline sequence) is a result of a single mutation giving rise to an NDT motif in the antibody sequence. Peptide mapping also provides a wealth of information on lower-abundance proteolytic fragments derived from the polyclonal component of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1 through IgG4) and both types of the light chain (λ and κ). The structural information reported in this work will be indispensable for understanding the molecular mechanism of VITT pathogenesis.

Antibodies against platelet factor 4 and the risk of cerebral venous sinus thrombosis in patients with vaccine-induced immune thrombotic thrombocytopenia.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenoviral vector-based vaccines against SARS-CoV-2. This syndrome is caused by antibodies against platelet factor 4 (PF4; CXCL4) that lead to platelet activation and is characterized by thrombocytopenia and thrombosis in unusual locations, including cerebral venous sinus thrombosis (CVST). VITT can be classified based on anti-PF4 antibodies properties in vitro: those that require PF4 to activate platelets (PF4-dependent) and those that can activate platelets without additional PF4 (PF4-independent) in the serotonin release assay. OBJECTIVES: We aim to characterize the relationship of VITT platelet-activating profiles with CVST. METHODS: We conducted a retrospective cohort study involving patients with confirmed VITT who were tested between March and June 2021. Data were collected with an anonymized form and cases were identified as VITT with high clinical suspicion according to platelet activation assays. Anti-PF4 antibody binding regions on PF4 were further characterized with alanine scanning mutagenesis. RESULTS: Of the patients with confirmed VITT (n = 39), 17 (43.6%) had PF4-dependent antibodies and 22 (56.4%) had PF4-independent antibodies. CVST occurred almost exclusively in PF4-independent patients (11 of 22 vs 1 of 17; P < .05). Additionally, PF4-independent antibodies bound to 2 distinct epitopes on PF4, the heparin-binding region and a site typical for heparin-induced thrombocytopenia antibodies, whereas PF4-dependent antibodies bound to only the heparin-binding region. CONCLUSION: These findings suggest that VITT antibodies that cause PF4-independent platelet activation represent a unique subset of patients more likely to be associated with CVST, possibly due to the 2 different types of anti-PF4 antibodies.

Investigation of anti-PF4 versus anti-PF4/heparin reactivity using fluid-phase enzyme immunoassay for 4 anti-PF4 disorders: classic heparin-induced thrombocytopenia (HIT), autoimmune HIT, vaccine-induced immune thrombotic thrombocytopenia, and spontaneous HIT.

BACKGROUND: Four platelet-activating anti-platelet factor 4 (PF4) disorders have been recognized: classic heparin-induced thrombocytopenia (cHIT), autoimmune heparin-induced thrombocytopenia (aHIT), spontaneous heparin-induced thrombocytopenia (SpHIT), and vaccine-induced immune thrombotic thrombocytopenia (VITT). All test immunoglobulin G (IgG) positive using solid-phase enzyme immunoassay (solid-EIA) against PF4/heparin (PF4/H) and/or PF4 alone. Fluid-phase EIA (fluid-EIA) should better discriminate between anti-PF4 and anti-PF4/H antibodies since conformationally altered PF4 bound to solid phase is avoided. OBJECTIVES: To compare anti-PF4 vs anti-PF4/H antibody profiles for anti-PF4 disorders using solid- and fluid-EIA. METHODS: We developed a novel fluid-EIA to measure anti-PF4 vs anti-PF4/H antibodies. RESULTS: Using fluid-EIA, 27 of 27 (100%) cHIT sera tested IgG positive with PF4/H, but only 4 of 27 (14.8%) tested positive against PF4 alone; all 27 exhibited heparin-enhanced binding. In contrast, 17 of 17 (100%) VITT sera tested IgG positive against PF4 alone, with markedly reduced binding against PF4/H; this distinct VITT antibody profile was not evident using solid-EIA. All 15 aHIT sera and all 11 SpHIT sera tested IgG positive against PF4 alone, with variable reactivity in PF4/H-EIA (heparin-enhanced binding in 14 of 15 and 10 of 11 aHIT and SpHIT sera, respectively). Remarkably, 1 SpHIT patient with a VITT-mimicking fluid-EIA profile (PF4 >> PF4/H) also clinically resembled patients with VITT (postviral cerebral vein/sinus thrombosis), with anti-PF4 reactivity correlating inversely with platelet count recovery; moreover, the single aHIT patient with a VITT-mimicking fluid-EIA profile also developed postviral cerebral vein/sinus thrombosis. CONCLUSION: cHIT and VITT sera showed opposite fluid-EIA profiles (cHIT: PF4/H >> PF4, with most testing negative against PF4 alone; VITT: PF4 >> PF4/H, with most testing negative against PF4/H). In contrast, all aHIT and SpHIT sera reacted against PF4 alone but with variable (usually enhanced) reactivity against PF4/H. VITT-mimicking clinical/serologic profiles occurred in only a minority of patients with SpHIT and aHIT.

Proceedings of the immune thrombocytopenia summit: new concepts in mechanisms, diagnosis, and management.

The inaugural McMaster Immune Thrombocytopenia (ITP) Summit was held virually in 2021. The objectives of the Summit were to recognize the difficulties in establishing the diagnosis of ITP and to understand gaps in current knowledge of ITP mechanisms that might lead to better diagnostic approaches and treatments. The half-day program consisted of virtual educational sessions targeting clinicians and basic scientists. The planning committee chose 8 topics to review that would cover current knowledge and inform future research priorities. In this report, we summarized the presentations delivered at the 2021 McMaster ITP Summit and the discussions. Based on the information presented at the Summit, the following research priorities were identified: 1) investigation of platelet production as a target for ITP treatments; 2) characterization of antigen processing and antigen presentation on platelets; 3) interaction between megakaryocytes and the immune system; 4) the role for ITP gene panels; 5) the need for better methods for platelet antibody testing; 6) the role of prediction models for diagnosis and prognosis; 7) new treatment strategies, including intensification of initial therapy; and 8) personalized treatment algorithms.

Molecular architecture and platelet-activating properties of small immune complexes assembled on intact heparin and their possible involvement in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin leading to a reduction in circulating platelets with an increased risk of thrombosis. It is precipitated by polymerized immune complexes consisting of pathogenic antibodies that recognize a small chemokine platelet factor 4 (PF4) bound to heparin, which trigger platelet activation and a hypercoagulable state. Characterization of these immune complexes is extremely challenging due to the enormous structural heterogeneity of such macromolecular assemblies and their constituents (especially heparin). We use native mass spectrometry to characterize small immune complexes formed by PF4, heparin and monoclonal HIT-specific antibodies. Up to three PF4 tetramers can be assembled on a heparin chain, consistent with the results of molecular modeling studies showing facile polyanion wrapping along the polycationic belt on the PF4 surface. Although these assemblies can accommodate a maximum of only two antibodies, the resulting immune complexes are capable of platelet activation despite their modest size. Taken together, these studies provide further insight into molecular mechanisms of HIT and other immune disorders where anti-PF4 antibodies play a central role.

Comparison of three dosing intervals for the primary vaccination of the SARS-CoV-2 mRNA Vaccine (BNT162b2) on magnitude, neutralization capacity and durability of the humoral immune response in health care workers: A prospective cohort study.

OBJECTIVES: The dosing interval of a primary vaccination series can significantly impact on vaccine immunogenicity and efficacy. The current study compared 3 dosing intervals for the primary vaccination series of the BNT162b2 mRNA COVID-19 vaccine, on humoral immune response and durability against SARS-CoV-2 ancestral and Beta variants up to 9 months post immunization. METHODS: Three groups of age- and sex-matched healthcare workers (HCW) who received 2 primary doses of BNT162b2 separated by 35-days, 35-42 days or >42-days were enrolled. Vaccine induced antibody titers at 3 weeks, 3 and 6-9 months post-second dose were assessed. RESULTS: There were 309 age- and sex-matched HCW (mean age 43 [sd 13], 58% females) enrolled. Anti-SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibody titers showed significant waning in levels beyond 35 days post first dose. The second dose induced a significant rise in antibody titers, which peaked at 3 weeks and then declined at variable rates across groups. The magnitude, consistency and durability of response was greater for anti-Spike than anti-RBD antibodies; and for IgG than IgA or IgM. Compared to the shorter schedules, a longer interval of >42 days offered the highest binding and neutralizing antibody titers against SARS-CoV-2 ancestral and Beta (B1.351) variants beyond 3 months post-vaccination. CONCLUSIONS: This is the first comprehensive study to compare 3 dosing intervals for the primary vaccination of BNT162b2 mRNA COVID-19 vaccine implemented in the real world. These findings suggest that delaying the second dose beyond 42 days can potentiate and prolong the humoral response against ancestral and Beta variants of SARS-CoV-2 up to 9 months post-vaccination.