Faculty Bibliography

The advent of COVID-19 vaccines will play a major role in helping to end the pandemic that has killed millions worldwide. Vaccine candidates have demonstrated robust humoral responses and have protected against infection. However, efficacy trials were focused on individuals with no prior exposure to SARS-CoV-2, and, as a result, little is known about immune responses induced by these mRNA vaccines in individuals who recovered from COVID-19. Here, we evaluated immune responses in 32 subjects who received two-dose BNT162b2 mRNA vaccination. In individuals naive to SARS-CoV-2, we observed robust increases in humoral and antigen-specific antibody-secreting cell (ASC) responses following each dose of vaccine, whereas individuals with prior exposure to SARS-CoV-2 demonstrated strong humoral and antigen-specific ASC responses to the first dose but muted responses to the second dose of the vaccine for the time points studied. These data highlight an important gap in our knowledge and may have major implications for how these vaccines should be used to prevent COVID-19.

The increasing prevalence of SARS-CoV-2 variants has raised concerns regarding possible decreases in vaccine efficacy. Here, neutralizing antibody titers elicited by mRNA-based and an adenoviral vector-based vaccine against variant pseudotyped viruses were compared. BNT162b2 and mRNA-1273-elicited antibodies showed modest neutralization resistance against Beta, Delta, Delta plus and Lambda variants whereas Ad26.COV2.S-elicited antibodies from a significant fraction of vaccinated individuals were of low neutralizing titer (IC ₅₀ <50). The data underscore the importance of surveillance for breakthrough infections that result in severe COVID-19 and suggest the benefit of a second immunization following Ad26.COV2.S to increase protection against the variants.

Antibody responses serve as the primary protection against SARS-CoV-2 infection through neutralization of viral entry into cells. We have developed a two-dimensional multiplex bead binding assay (2D-MBBA) that quantifies multiple antibody isotypes against multiple antigens from a single measurement. Here, we applied our assay to profile IgG, IgM and IgA levels against the spike antigen, its receptor-binding domain and natural and designed mutants. Machine learning algorithms trained on the 2D-MBBA data substantially improve the prediction of neutralization capacity against the authentic SARS-CoV-2 virus of serum samples of convalescent patients. The algorithms also helped identify a set of antibody isotypeâ€"antigen datasets that contributed to the prediction, which included those targeting regions outside the receptor-binding interface of the spike protein. We applied the assay to profile samples from vaccinated, immune-compromised patients, which revealed differences in the antibody profiles between convalescent and vaccinated samples. Our approach can rapidly provide deep antibody profiles and neutralization prediction from essentially a drop of blood without the need of BSL-3 access and provides insights into the nature of neutralizing antibodies. It may be further developed for evaluating neutralizing capacity for new variants and future pathogens.

DNA sequence analysis recently identified the novel SARS-CoV-2 variant B.1.526 that is spreading at an alarming rate in the New York City area. Two versions of the variant were identified, both with the prevalent D614G mutation in the spike protein, together with four novel point mutations and with an E484K or S477N mutation in the receptor-binding domain, raising concerns of possible resistance to vaccine-elicited and therapeutic antibodies. We report that convalescent-phase sera and vaccine-elicited antibodies retain full neutralizing titer against the S477N B.1.526 variant and neutralize the E484K version with a modest 3.5-fold decrease in titer compared to D614G. The E484K version was neutralized with a 12-fold decrease in titer by the REGN10933 monoclonal antibody, but the combination cocktail with REGN10987 was fully active. The findings suggest that current vaccines and Regeneron therapeutic monoclonal antibodies will remain protective against the B.1.526 variants. The findings further support the value of widespread vaccination. IMPORTANCE A novel SARS-CoV-2 variant termed B.1.526 was recently identified in New York City and has been found to be spreading at an alarming rate. The variant has mutations in its spike protein that might allow it to escape neutralization by vaccine-elicited antibodies and might cause monoclonal antibody therapy for COVID-19 to be less successful. We report here that these fears are not substantiated; convalescent-phase sera and vaccine-elicited antibodies neutralized the B.1.526 variant. One of the Regeneron therapeutic monoclonal antibodies was less effective against the B.1.526 (E484K) variant but the two-antibody combination cocktail was fully active. The findings should assuage concerns that current vaccines will be ineffective against the B.1.526 (E484K) variant and suggest the importance of continued widespread vaccination.

Infections and inflammation are profoundly influenced by the extracellular matrix (ECM), but their molecular underpinnings are ill defined. Here, we demonstrate that lumican, an ECM protein normally associated with collagens, is elevated in sepsis patients' blood, while lumican-null mice resolve polymicrobial sepsis poorly, with reduced bacterial clearance and greater body weight loss. Secreted by activated fibroblasts, lumican promotes Toll-like receptor (TLR) 4 response to bacterial lipopolysaccharides (LPS) but restricts nucleic acid-specific TLR9 in macrophages and dendritic cells. The underlying mechanism involves lumican attachment to the common TLR coreceptor CD14 and caveolin 1 (Cav1) in lipid rafts on immune cell surfaces via two epitopes, which may be cryptic in collagen-associated lumican. The Cav1 binding epitope alone is sufficient for cell surface enrichment of Cav1, while both are required for lumican to increase cell surface TLR4, CD14, and proinflammatory cytokines in response to LPS. Endocytosed lumican colocalizes with TLR4 and LPS and promotes endosomal induction of type I interferons. Lumican-null macrophages show elevated TLR9 in signal-permissive endolysosomes and increased response, while wild types show lumican colocalization with CpG DNA but not TLR9, consistent with a ligand sequestering, restrictive role for lumican in TLR9 signaling. In vitro, lumican competes with CD14 to bind CpG DNA; biglycan, a lumican paralog, also binds CpG DNA and suppresses TLR9 response. Thus, lumican and other ECM proteins, synthesized de novo or released from collagen association during ECM remodeling, may be internalized by immune cells to regulate their transcriptional programs and effector responses that may be harnessed in future therapeutics.

The increasing prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with spike protein mutations raises concerns that antibodies elicited by natural infection or vaccination and therapeutic monoclonal antibodies will become less effective. We show that convalescent-phase sera neutralize pseudotyped viruses with the B.1.1.7, B.1.351, B.1.1.248, COH.20G/677H, 20A.EU2, and mink cluster 5 spike proteins with only a minor loss in titer. Similarly, antibodies elicited by Pfizer BNT162b2 vaccination neutralized B.1.351 and B.1.1.248 with only a 3-fold decrease in titer, an effect attributable to E484K. Analysis of the Regeneron monoclonal antibodies REGN10933 and REGN10987 showed that REGN10933 has lost neutralizing activity against the B.1.351 and B.1.1.248 pseudotyped viruses, and the cocktail is 9- to 15-fold decreased in titer. These findings suggest that antibodies elicited by natural infection and by the Pfizer vaccine will maintain protection against the B.1.1.7, B.1.351, and B.1.1.248 variants but that monoclonal antibody therapy may be less effective for patients infected with B.1.351 or B.1.1.248 SARS-CoV-2. IMPORTANCE The rapid evolution of SARS-CoV-2 variants has raised concerns with regard to their potential to escape from vaccine-elicited antibodies and anti-spike protein monoclonal antibodies. We report here on an analysis of sera from recovered patients and vaccinated individuals and on neutralization by Regeneron therapeutic monoclonal antibodies. Overall, the variants were neutralized nearly as well as the wild-type pseudotyped virus. The B.1.351 variant was somewhat resistant to vaccine-elicited antibodies but was still readily neutralized. One of the two Regeneron therapeutic monoclonal antibodies seems to have lost most of its activity against the B.1.351 variant, raising concerns that the combination therapy might be less effective for some patients. The findings should alleviate concerns that vaccines will become ineffective but suggest the importance of continued surveillance for potential new variants.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics which may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days, and clearance of 0.22 mL/hr/kg, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study Day 6 following viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

Objective/UNASSIGNED:To investigate the humoral and cellular immune response to mRNA COVID-19 vaccines in patients with immune-mediated inflammatory diseases (IMIDs) on immunomodulatory treatment. Methods/UNASSIGNED:Established patients at NYU Langone Health with IMID (n=51) receiving the BNT162b2 mRNA vaccination were assessed at baseline and after second immunization. Healthy subjects served as controls (n=26). IgG antibody responses to the spike protein were analyzed for humoral response. Cellular immune response to SARS-CoV-2 was further analyzed using high-parameter spectral flow cytometry. A second independent, validation cohort of controls (n=182) and patients with IMID (n=31) from Erlangen, Germany were also analyzed for humoral immune response. Results/UNASSIGNED:Although healthy subjects (n=208) and IMID patients on biologic treatments (mostly on TNF blockers, n=37) demonstrate robust antibody responses (over 90%), those patients with IMID on background methotrexate (n=45) achieve an adequate response in only 62.2% of cases. Similarly, IMID patients do not demonstrate an increase in CD8+ T cell activation after vaccination. Conclusions/UNASSIGNED:In two independent cohorts of IMID patients, methotrexate, a widely used immunomodulator for the treatment of several IMIDs, adversely affected humoral and cellular immune response to COVID-19 mRNA vaccines. Although precise cut offs for immunogenicity that correlate with vaccine efficacy are yet to be established, our findings suggest that different strategies may need to be explored in patients with IMID taking methotrexate to increase the chances of immunization efficacy against SARS-CoV-2 as has been demonstrated for augmenting immunogenicity to other viral vaccines. KEY MESSAGES/UNASSIGNED:These results suggest that patients on methotrexate may need alternate vaccination strategies such as additional doses of vaccine, dose modification of methotrexate, or even a temporary discontinuation of this drug. Further studies will be required to explore the effect of these approaches on mRNA vaccine immunogenicity.

DNA sequence analysis recently identified the novel SARS-CoV-2 variant B.1.526 that is spreading at an alarming rate in the New York City area. Two versions of the variant were identified, both with the prevalent D614G mutation in the spike protein together with four novel point mutations and with an E484K or S477N mutation in the receptor binding domain, raising concerns of possible resistance to vaccine-elicited and therapeutic antibodies. We report that convalescent sera and vaccine-elicited antibodies retain full neutralizing titer against the S477N B.1.526 variant and neutralize the E484K version with a modest 3.5-fold decrease in titer as compared to D614G. The E484K version was neutralized with a 12-fold decrease in titer by the REGN10933 monoclonal antibody but the combination cocktail with REGN10987 was fully active. The findings suggest that current vaccines and therapeutic monoclonal antibodies will remain protective against the B.1.526 variants. The findings further support the value of wide-spread vaccination.

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization.