Publications by authors named "Michel C Nussenzweig"

329 Publications

Germinal center-dependent and -independent memory B cells produced throughout the immune response.

J Exp Med 2021 Aug 9;218(8). Epub 2021 Jun 9.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY.

Memory B cells comprise a heterogenous group of cells that differ in origin and phenotype. During the early phases of the immune response, activated B cells can differentiate into IgM-expressing memory cells, short-lived plasma cells, or seed germinal centers (GCs). The memory compartment is subsequently enriched by B cells that have been through several rounds of division and selection in the GC. Here, we report on the use of an unbiased lineage-tracking approach to explore the origins and properties of memory B cell subsets in mice with an intact immune system. We find that activated B cells continue to differentiate into memory B cells throughout the immune response. When defined on the basis of their origins, the memory B cells originating from activated B cells or GCs differ in isotype and overall gene expression, somatic hypermutation, and their affinity for antigen.
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http://dx.doi.org/10.1084/jem.20202489DOI Listing
August 2021

Naturally enhanced neutralizing breadth to SARS-CoV-2 after one year.

bioRxiv 2021 Jun 2. Epub 2021 Jun 2.

Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies . Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines . In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naïve individuals . The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern . In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.
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http://dx.doi.org/10.1101/2021.05.07.443175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183008PMC
June 2021

Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants.

Nature 2021 Jun 7. Epub 2021 Jun 7.

Lymphocyte Nuclear Biology, NIAMS, NIH, Bethesda, MD, 20892, USA.

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. While many vaccines have been deployed to date, the continual evolution of the viral receptor-binding domain (RBD) has challenged their efficacy. In particular, emerging variants B.1.1.7 (U.K.), B.1.351 (South Africa) and P.1 (Brazil) have compromised convalescent sera and immunotherapies that received emergency use authorization. One potential alternative to avert viral escape is the use of camelid VHHs or nanobodies, which can recognize epitopes often inaccessible to conventional antibodies. Here, we isolate anti-RBD nanobodies from llamas and "nanomice" we engineered to produce VHHs cloned from alpacas, dromedaries and camels. We identified two sets of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD-ACE2 interface and fails to neutralize variants carrying E484K or N501Y substitutions. Notably however, group 2 nanobodies retain full neutralization activity against variants when expressed as homotrimers, rivaling the most potent antibodies produced to date against SARS-CoV-2. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2 binding domain, and recognition of conserved epitopes largely inaccessible to human antibodies. Therefore, while new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.
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http://dx.doi.org/10.1038/s41586-021-03676-zDOI Listing
June 2021

Broadly neutralizing antibody-mediated protection of macaques against repeated intravenous exposures to simian-human immunodeficiency virus.

AIDS 2021 May 5. Epub 2021 May 5.

Laboratory Branch, Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, GA Laboratory of Molecular Immunology, The Rockefeller University, New York, NY Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA Howard Hughes Medical Institute, The Rockefeller University, New York, NY.

Objective: The opioid epidemic has increased parentally acquired HIV infection. To inform the development of a long-acting prevention strategy, we evaluated the protective efficacy of broadly neutralizing antibodies (bNAbs) against intravenous SHIV infection in macaques.

Design: Five cynomolgus macaques were injected once subcutaneously with 10-1074 and 3BNC117 (10 mg each kg-1) and were repeatedly challenged intravenously once weekly with SHIVAD8-EO (130 TCID50), until infection was confirmed via plasma viral load assay. Two control macaques, which received no antibody, were challenged identically.

Methods: Plasma viremia was monitored via RT-qPCR assay. bNAb concentrations were determined longitudinally in plasma samples via TZM-bl neutralization assays using virions pseudotyped with 10-1074-sensitive (X2088_c9) or 3BNC117-sensitive (Q769.d22) HIV envelope proteins.

Results: Passively immunized macaques were protected against a median of five weekly intravenous SHIV challenges, as compared to untreated controls, which were infected following a single challenge. Of the two bNAbs, 10-1074 exhibited relatively longer persistence in vivo. The median plasma level of 10-1074 at SHIV breakthrough was 1.1 μg ml-1 (range: 0.6-1.6 μg ml-1), whereas 3BNC117 was undetectable. Probit modeling estimated that 6.6 μg ml-1 of 10-1074 in plasma corresponded to a 99% reduction in per-challenge infection probability, as compared to controls.

Conclusions: Significant protection against repeated intravenous SHIV challenges was observed following administration of 10-1074 and 3BNC117 and was due primarily to 10-1074. Our findings extend preclinical studies of bNAb-mediated protection against mucosal SHIV acquisition and support the possibility that intermittent subcutaneous injections of 10-1074 could serve as long-acting pre-exposure prophylaxis for persons who inject drugs.
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http://dx.doi.org/10.1097/QAD.0000000000002934DOI Listing
May 2021

Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies.

bioRxiv 2021 Apr 26. Epub 2021 Apr 26.

Many anti-SARS-CoV-2 neutralizing antibodies target the ACE2-binding site on viral spike receptor-binding domains (RBDs). The most potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly-emergent zoonotic sarbecoviruses and variants, but usually show only weak neutralization potencies. We characterized two class 4 anti-RBD antibodies derived from COVID-19 donors that exhibited broad recognition and potent neutralization of zoonotic coronavirus and SARS-CoV-2 variants. C118-RBD and C022-RBD structures revealed CDRH3 mainchain H-bond interactions that extended an RBD β-sheet, thus reducing sensitivity to RBD sidechain changes, and epitopes that extended from the cryptic epitope to occlude ACE2 binding. A C118-spike trimer structure revealed rotated RBDs to allow cryptic epitope access and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.
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http://dx.doi.org/10.1101/2021.04.23.441195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095199PMC
April 2021

TOP-Plus is a Versatile Biosensor Platform for Monitoring SARS-CoV-2 Antibody Durability.

Clin Chem 2021 Apr 29. Epub 2021 Apr 29.

Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA.

Background: Low initial SARS-CoV-2 antibody titers dropping to undetectable levels within months after infection have raised concerns over long term immunity. Both the antibody levels and avidity of the antibody-antigen interaction should be examined to understand the quality of the antibody response.

Methods: A testing-on-a-probe "plus" panel (TOP-Plus) was developed, which included a newly developed avidity assay built into the previously described SARS-CoV-2 TOP assays that measured total antibody (TAb), surrogate neutralizing antibody (SNAb), IgM and IgG on a versatile biosensor platform. TAb and SNAb levels were compared with avidity in previously infected individuals at 1.3 and 6.2 months post-infection in paired samples from 80 COVID-19 patients. Sera from SARS-CoV-2 vaccinated individuals were also evaluated for antibody avidity.

Results: The newly designed avidity assay in this TOP panel correlated well with a reference Bio-Layer Interferometry avidity assay (r=0.88). The imprecision of the TOP avidity assay was less than 10%. Although TAb and neutralization activity (by SNAb) decreased between 1.3 and 6.2 months post-infection, the antibody avidity increased significantly (P < 0.0001). Antibody avidity in 10 SARS-CoV-2 vaccinated individuals (median 28 days post-vaccination) was comparable to the measured antibody avidity in infected individuals (median 26 days post-infection).

Conclusion: This highly precise and versatile TOP-Plus panel with the ability to measure SARS-CoV-2 TAb, SNAb, IgG and IgM antibody levels and avidity of individual sera on one sensor can become a valuable asset in monitoring not only SARS-CoV-2-infected patients, but also the status of individuals' COVID-19 vaccination response.
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http://dx.doi.org/10.1093/clinchem/hvab069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8135537PMC
April 2021

Detection and characterization of the SARS-CoV-2 lineage B.1.526 in New York.

bioRxiv 2021 Apr 22. Epub 2021 Apr 22.

Wide-scale SARS-CoV-2 genome sequencing is critical to tracking viral evolution during the ongoing pandemic. Variants first detected in the United Kingdom, South Africa, and Brazil have spread to multiple countries. We developed the software tool, Variant Database (VDB), for quickly examining the changing landscape of spike mutations. Using VDB, we detected an emerging lineage of SARS-CoV-2 in the New York region that shares mutations with previously reported variants. The most common sets of spike mutations in this lineage (now designated as B.1.526) are L5F, T95I, D253G, E484K or S477N, D614G, and A701V. This lineage was first sequenced in late November 2020 when it represented <1% of sequenced coronavirus genomes that were collected in New York City (NYC). By February 2021, genomes from this lineage accounted for 32% of 3288 sequenced genomes from NYC specimens. Phylodynamic inference confirmed the rapid growth of the B.1.526 lineage in NYC, notably the sub-clade defined by the spike mutation E484K, which has outpaced the growth of other variants in NYC. Pseudovirus neutralization experiments demonstrated that B.1.526 spike mutations adversely affect the neutralization titer of convalescent and vaccinee plasma, indicating the public health importance of this lineage.
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http://dx.doi.org/10.1101/2021.02.14.431043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8077570PMC
April 2021

Vaccine Breakthrough Infections with SARS-CoV-2 Variants.

N Engl J Med 2021 06 21;384(23):2212-2218. Epub 2021 Apr 21.

From the Laboratory of Molecular Neuro-oncology (E.H., C.H., Y.S., N.E.B., M.B., E.G.C., R.B.D.), the Laboratory of Molecular Immunology (D.J.S.-B., C.G., M.C.N.), the Laboratory of Human Genetics and Genomics (R.L.), the Laboratory of Retrovirology (J.D., F.M., T.H., P.D.B.), and the Howard Hughes Medical Institute (M.C.N., P.D.B., R.B.D.), Rockefeller University, New York.

Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of clinical concern. In a cohort of 417 persons who had received the second dose of BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccine at least 2 weeks previously, we identified 2 women with vaccine breakthrough infection. Despite evidence of vaccine efficacy in both women, symptoms of coronavirus disease 2019 developed, and they tested positive for SARS-CoV-2 by polymerase-chain-reaction testing. Viral sequencing revealed variants of likely clinical importance, including E484K in 1 woman and three mutations (T95I, del142-144, and D614G) in both. These observations indicate a potential risk of illness after successful vaccination and subsequent infection with variant virus, and they provide support for continued efforts to prevent and diagnose infection and to characterize variants in vaccinated persons. (Funded by the National Institutes of Health and others.).
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http://dx.doi.org/10.1056/NEJMoa2105000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8117968PMC
June 2021

Broad and potent neutralizing human antibodies to tick-borne flaviviruses protect mice from disease.

J Exp Med 2021 May;218(5)

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY.

Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV.
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http://dx.doi.org/10.1084/jem.20210236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040517PMC
May 2021

Sequencing, cloning, and antigen binding analysis of monoclonal antibodies isolated from single mouse B cells.

STAR Protoc 2021 Jun 15;2(2):100389. Epub 2021 Mar 15.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.

The analysis of B cell receptors (BCR) from single B cells is crucial to understanding humoral immune responses. Here, we describe a protocol for the sequencing, cloning, and characterization of antibody genes that encode BCRs. We used this method to analyze the BCRs of different mouse B cell populations for somatic hypermutations, clonal and phylogenic relationships, and their affinity for cognate antigen. For complete details on the use and execution of this protocol, please refer to Viant et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2021.100389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985702PMC
June 2021

Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice.

Nature 2021 05 25;593(7859):424-428. Epub 2021 Mar 25.

European Commission, Joint Research Centre, Ispra, Italy.

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-19. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-19. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.
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http://dx.doi.org/10.1038/s41586-021-03461-yDOI Listing
May 2021

Development of potency, breadth and resilience to viral escape mutations in SARS-CoV-2 neutralizing antibodies.

bioRxiv 2021 Mar 8. Epub 2021 Mar 8.

Antibodies elicited in response to infection undergo somatic mutation in germinal centers that can result in higher affinity for the cognate antigen. To determine the effects of somatic mutation on the properties of SARS-CoV-2 spike receptor-binding domain (RBD)-specific antibodies, we analyzed six independent antibody lineages. As well as increased neutralization potency, antibody evolution changed pathways for acquisition of resistance and, in some cases, restricted the range of neutralization escape options. For some antibodies, maturation apparently imposed a requirement for multiple spike mutations to enable escape. For certain antibody lineages, maturation enabled neutralization of circulating SARS-CoV-2 variants of concern and heterologous sarbecoviruses. Antibody-antigen structures revealed that these properties resulted from substitutions that allowed additional variability at the interface with the RBD. These findings suggest that increasing antibody diversity through prolonged or repeated antigen exposure may improve protection against diversifying SARS-CoV-2 populations, and perhaps against other pandemic threat coronaviruses.
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http://dx.doi.org/10.1101/2021.03.07.434227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987023PMC
March 2021

Mutational escape from the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of antibodies.

bioRxiv 2021 Mar 18. Epub 2021 Mar 18.

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasma, including plasma from individuals from whom some of the antibodies were isolated. The plasma-escape maps most closely resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is dominated by a single class of antibodies targeting an epitope that is already undergoing rapid evolution.
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http://dx.doi.org/10.1101/2021.03.17.435863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987015PMC
March 2021

Multimeric nanobodies from camelid engineered mice and llamas potently neutralize SARS-CoV-2 variants.

bioRxiv 2021 Mar 4. Epub 2021 Mar 4.

Since the start of the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 2 million deaths worldwide. Multiple vaccines have been deployed to date, but the continual evolution of the viral receptor-binding domain (RBD) has recently challenged their efficacy. In particular, SARS-CoV-2 variants originating in the U.K. (B.1.1.7), South Africa (B.1.351) and New York (B.1.526) have reduced neutralization activity from convalescent sera and compromised the efficacy of antibody cocktails that received emergency use authorization. Whereas vaccines can be updated periodically to account for emerging variants, complementary strategies are urgently needed to avert viral escape. One potential alternative is the use of camelid VHHs (also known as nanobodies), which due to their small size can recognize protein crevices that are inaccessible to conventional antibodies. Here, we isolate anti-RBD nanobodies from llamas and "nanomice" we engineered to produce VHHs cloned from alpacas, dromedaries and camels. Through binding assays and cryo-electron microscopy, we identified two sets of highly neutralizing nanobodies. The first group expresses VHHs that circumvent RBD antigenic drift by recognizing a region outside the ACE2-binding site that is conserved in coronaviruses but is not typically targeted by monoclonal antibodies. The second group is almost exclusively focused to the RBD-ACE2 interface and fails to neutralize pseudoviruses carrying the E484K or N501Y substitutions. Notably however, they do neutralize the RBD variants when expressed as homotrimers, rivaling the most potent antibodies produced to date against SARS-CoV-2. These findings demonstrate that multivalent nanobodies overcome SARS-CoV-2 variant mutations through two separate mechanisms: enhanced avidity for the ACE2 binding domain, and recognition of conserved epitopes largely inaccessible to human antibodies. Therefore, while new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.
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http://dx.doi.org/10.1101/2021.03.04.433768DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941635PMC
March 2021

A clinical trial of non-invasive imaging with an anti-HIV antibody labelled with copper-64 in people living with HIV and uninfected controls.

EBioMedicine 2021 Mar 25;65:103252. Epub 2021 Feb 25.

Department of Molecular Imaging and Therapy, Austin Health, and University of Melbourne, Melbourne, Australia; Tumour Targeting Laboratory, Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Melbourne, Australia.

Background: A research priority in finding a cure for HIV is to establish methods to accurately locate and quantify where and how HIV persists in people living with HIV (PLWH) receiving suppressive antiretroviral therapy (ART). Infusing copper-64 (Cu) radiolabelled broadly neutralising antibodies targeting HIV envelope (Env) with CT scan and positron emission tomography (PET) identified HIV Env in tissues in SIV infected non-human primates . We aimed to determine if a similar approach was effective in people living with HIV (PLWH).

Methods: Unmodified 3BNC117 was compared with 3BNC117 bound to the chelator MeCOSar and Cu (Cu-3BNC117) in vitro to assess binding and neutralization. In a clinical trial Cu-3BNC117 was infused into HIV uninfected (Group 1), HIV infected and viremic (viral load, VL >1000 c/mL; Group 2) and HIV infected aviremic (VL <20 c/mL; Group 3) participants using two dosing strategies: high protein (3mg/kg unlabeled 3BNC117 combined with <5mg Cu-3BNC117) and trace (<5mg Cu-3BNC117 only). All participants were screened for 3BNC117 sensitivity from virus obtained from viral outgrowth. Magnetic resonance imaging (MRI)/PET and pharmacokinetic assessments (ELISA for serum 3BNC117 concentrations and gamma counting for Cu) were performed 1, 24- and 48-hours post dosing. The trial (clincialtrials.gov NCT03063788) primary endpoint was comparison of PET standard uptake values (SUVs) in regions of interest (e.g lymph node groups and gastrointestinal tract).

Findings: Comparison of unmodified and modified 3BNC117 in vitro demonstrated no difference in HIV binding or neutralisation. 17 individuals were enrolled of which 12 were dosed including Group 1 (n=4, 2 high protein, 2 trace dose), Group 2 (n=6, 2 high protein, 4 trace) and Group 3 (n=2, trace only). HIV+ participants had a mean CD4 of 574 cells/microL and mean age 43 years. There were no drug related adverse effects and no differences in tissue uptake in regions of interest (e.g lymph node gut, pharynx) between the 3 groups. In the high protein dosing group, serum concentrations of 3BNC117 and gamma counts were highly correlated demonstrating that Cu-3BNC117 remained intact in vivo.

Interpretation: In PLWH on or off ART, the intervention of infusing Cu-3BNC117 and MRI/PET imaging over 48 hours, was unable to detect HIV-1 env expression in vivo. Future studies should investigate alternative radiolabels such as zirconium which have a longer half-life in vivo.

Funding: Funded by the Alfred Foundation, The Australian Centre for HIV and Hepatitis Virology Research with additional support from the Division of AIDS, National Institute of Allergy and Infectious Disease, US National Institutes of Health (USAI126611). JHM and SRL are supported by the Australian National Health and Medical Research Council.
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http://dx.doi.org/10.1016/j.ebiom.2021.103252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921458PMC
March 2021

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

Nature 2021 04 10;592(7855):616-622. Epub 2021 Feb 10.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.

Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-2. Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection. However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors. However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.
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http://dx.doi.org/10.1038/s41586-021-03324-6DOI Listing
April 2021

TOP-Plus is a Versatile Biosensor Platform for Monitoring SARS-CoV-2 Antibody Durability.

medRxiv 2021 Feb 8. Epub 2021 Feb 8.

Background: There is a concern that low initial SARS-CoV-2 antibody titers in individuals may drop to undetectable levels within months after infection. Although this may raise concerns over long term immunity, both the antibody levels and avidity of the antibody-antigen interaction should be examined to understand the quality of the antibody response.

Methods: A testing-on-a-probe-plus panel (TOP-Plus) was developed, which included a newly developed avidity assay built into the previously described SARS-CoV-2 TOP assays that measured total antibody (TAb), surrogate neutralizing antibody (SNAb), IgM and IgG on a versatile biosensor platform. TAb and SNAb levels were compared with avidity in previously infected individuals at 1.3 and 6.2 months post-infection in paired samples from 80 COVID-19 patients.

Results: The newly designed avidity assay in this TOP panel correlated well with a reference Bio-Layer Interferometry avidity assay (R=0.88). The imprecision of the TOP avidity assay was less than 9%. Although TAb and neutralization activity (by SNAb) decreased between 1.3 and 6.2 months post infection, the antibody avidity increased significantly (P < 0.0001).
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http://dx.doi.org/10.1101/2021.02.03.21251089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872385PMC
February 2021

Dynamic regulation of T selection during the germinal centre reaction.

Nature 2021 Mar 3;591(7850):458-463. Epub 2021 Feb 3.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY, USA.

The germinal centre is a dynamic microenvironment in which B cells that express high-affinity antibody variants produced by somatic hypermutation are selected for clonal expansion by limiting the numbers of T follicular helper cells. Although much is known about the mechanisms that control the selection of B cells in the germinal centre, far less is understood about the clonal behaviour of the T follicular helper cells that help to regulate this process. Here we report on the dynamic behaviour of T follicular helper cell clones during the germinal centre reaction. We find that, similar to germinal centre B cells, T follicular helper cells undergo antigen-dependent selection throughout the germinal centre reaction that results in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal centre leads to increased division of T follicular helper cells. Competition between T follicular helper cell clones is mediated by the affinity of T cell receptors for peptide-major-histocompatibility-complex ligands. T cells that preferentially expand in the germinal centre show increased expression of genes downstream of the T cell receptor, such as those required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to marked remodelling of the functional T follicular helper cell repertoire during the germinal centre reaction.
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http://dx.doi.org/10.1038/s41586-021-03187-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979475PMC
March 2021

Persistent cellular immunity to SARS-CoV-2 infection.

J Exp Med 2021 04;218(4)

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY.

SARS-CoV-2 is responsible for an ongoing pandemic that has affected millions of individuals around the globe. To gain further understanding of the immune response in recovered individuals, we measured T cell responses in paired samples obtained an average of 1.3 and 6.1 mo after infection from 41 individuals. The data indicate that recovered individuals show persistent polyfunctional SARS-CoV-2 antigen-specific memory that could contribute to rapid recall responses. Recovered individuals also show enduring alterations in relative overall numbers of CD4+ and CD8+ memory T cells, including expression of activation/exhaustion markers, and cell division.
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http://dx.doi.org/10.1084/jem.20202515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845919PMC
April 2021

Early therapy with remdesivir and antibody combinations improves COVID-19 disease in mice.

bioRxiv 2021 Jan 28. Epub 2021 Jan 28.

Improving the standard of clinical care for coronavirus disease 2019 (COVID-19) is a global health priority. Small molecule antivirals like remdesivir (RDV) and biologics such as human monoclonal antibodies (mAb) have demonstrated therapeutic efficacy against SARS-CoV-2, the causative agent of COVID-19. However, the efficacy of single agent therapies has not been comprehensively defined over the time course of infection and it is not known if combination RDV/mAb will improve outcomes over single agent therapies. In kinetic studies in a mouse-adapted SARS-CoV-2 pathogenesis model, we show that single-agent therapies exert potent antiviral effects even when initiated relatively late after infection, but their efficacy is diminished as a function of time. RDV and a cocktail of two mAbs in combination provided improved outcomes compared to single agents alone extending the therapeutic window of intervention with less weight loss, decreased virus lung titers, reduced acute lung injury, and improved pulmonary function. Overall, we demonstrate that direct-acting antivirals combined with potent mAb can improve outcomes over single agents alone in animal models of COVID-19 thus providing a rationale for the coupling of therapies with disparate modalities to extend the therapeutic window of treatment.
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http://dx.doi.org/10.1101/2021.01.27.428478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7852229PMC
January 2021

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants.

bioRxiv 2021 Jan 19. Epub 2021 Jan 19.

To date severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected nearly 100 million individuals resulting in over two million deaths. Many vaccines are being deployed to prevent coronavirus disease-2019 (COVID-19) including two novel mRNA-based vaccines . These vaccines elicit neutralizing antibodies and appear to be safe and effective, but the precise nature of the elicited antibodies is not known . Here we report on the antibody and memory B cell responses in a cohort of 20 volunteers who received either the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccines. Consistent with prior reports, 8 weeks after the second vaccine injection volunteers showed high levels of IgM, and IgG anti-SARS-CoV-2 spike protein (S), receptor binding domain (RBD) binding titers . Moreover, the plasma neutralizing activity, and the relative numbers of RBD-specific memory B cells were equivalent to individuals who recovered from natural infection . However, activity against SARS-CoV-2 variants encoding E484K or N501Y or the K417N:E484K:N501Y combination was reduced by a small but significant margin. Consistent with these findings, vaccine-elicited monoclonal antibodies (mAbs) potently neutralize SARS-CoV-2, targeting a number of different RBD epitopes epitopes in common with mAbs isolated from infected donors. Structural analyses of mAbs complexed with S trimer suggest that vaccine- and virus-encoded S adopts similar conformations to induce equivalent anti-RBD antibodies. However, neutralization by 14 of the 17 most potent mAbs tested was reduced or abolished by either K417N, or E484K, or N501Y mutations. Notably, the same mutations were selected when recombinant vesicular stomatitis virus (rVSV)/SARS-CoV-2 S was cultured in the presence of the vaccine elicited mAbs. Taken together the results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid potential loss of clinical efficacy.
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http://dx.doi.org/10.1101/2021.01.15.426911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836122PMC
January 2021

Bispecific antibody prevents SARS-CoV-2 escape and protects mice from disease.

bioRxiv 2021 Jan 22. Epub 2021 Jan 22.

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19) . We developed a bispecific, IgG1-like molecule based on two antibodies derived from COVID-19 convalescent donors, C121 and C135 . CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, completely prevents S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 recognizes a broad panel of RBD variants and neutralizes SARS-CoV-2 and the escape mutants generated by the single monoclonals at sub-nanomolar concentrations. In a novel model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.
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http://dx.doi.org/10.1101/2021.01.22.427567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836104PMC
January 2021

Evolution of antibody immunity to SARS-CoV-2.

Nature 2021 03 18;591(7851):639-644. Epub 2021 Jan 18.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY, USA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected 78 million individuals and is responsible for over 1.7 million deaths to date. Infection is associated with the development of variable levels of antibodies with neutralizing activity, which can protect against infection in animal models. Antibody levels decrease with time, but, to our knowledge, the nature and quality of the memory B cells that would be required to produce antibodies upon reinfection has not been examined. Here we report on the humoral memory response in a cohort of 87 individuals assessed at 1.3 and 6.2 months after infection with SARS-CoV-2. We find that titres of IgM and IgG antibodies against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 decrease significantly over this time period, with IgA being less affected. Concurrently, neutralizing activity in plasma decreases by fivefold in pseudotype virus assays. By contrast, the number of RBD-specific memory B cells remains unchanged at 6.2 months after infection. Memory B cells display clonal turnover after 6.2 months, and the antibodies that they express have greater somatic hypermutation, resistance to RBD mutations and increased potency, indicative of continued evolution of the humoral response. Immunofluorescence and PCR analyses of intestinal biopsies obtained from asymptomatic individuals at 4 months after the onset of coronavirus disease 2019 (COVID-19) revealed the persistence of SARS-CoV-2 nucleic acids and immunoreactivity in the small bowel of 7 out of 14 individuals. We conclude that the memory B cell response to SARS-CoV-2 evolves between 1.3 and 6.2 months after infection in a manner that is consistent with antigen persistence.
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http://dx.doi.org/10.1038/s41586-021-03207-wDOI Listing
March 2021

Heightened resistance to host type 1 interferons characterizes HIV-1 at transmission and after antiretroviral therapy interruption.

Sci Transl Med 2021 Jan;13(576)

Laboratory of Molecular Immunology, Rockefeller University, New York, NY 10065, USA.

Type 1 interferons (IFN-I) are potent innate antiviral effectors that constrain HIV-1 transmission. However, harnessing these cytokines for HIV-1 cure strategies has been hampered by an incomplete understanding of their antiviral activities at later stages of infection. Here, we characterized the IFN-I sensitivity of 500 clonally derived HIV-1 isolates from the plasma and CD4 T cells of 26 individuals sampled longitudinally after transmission or after antiretroviral therapy (ART) and analytical treatment interruption. We determined the concentration of IFNα2 and IFNβ that reduced viral replication in vitro by 50% (IC) and found consistent changes in the sensitivity of HIV-1 to IFN-I inhibition both across individuals and over time. Resistance of HIV-1 isolates to IFN-I was uniformly high during acute infection, decreased in all individuals in the first year after infection, was reacquired concomitant with CD4 T cell loss, and remained elevated in individuals with accelerated disease. HIV-1 isolates obtained by viral outgrowth during suppressive ART were relatively IFN-I sensitive, resembling viruses circulating just before ART initiation. However, viruses that rebounded after treatment interruption displayed the highest degree of IFNα2 and IFNβ resistance observed at any time during the infection course. These findings indicate a dynamic interplay between host innate responses and the evolving HIV-1 quasispecies, with the relative contribution of IFN-I to HIV-1 control affected by both ART and analytical treatment interruption. Although elevated at transmission, host innate pressures are the highest during viral rebound, limiting the viruses that successfully become reactivated from latency to those that are IFN-I resistant.
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http://dx.doi.org/10.1126/scitranslmed.abd8179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923595PMC
January 2021

Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice.

Science 2021 02 12;371(6530):735-741. Epub 2021 Jan 12.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic RBD nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs relative to sera from immunizations with homotypic SARS-CoV-2-RBD nanoparticles or COVID-19 convalescent human plasmas. Moreover, after priming, sera from mosaic RBD-immunized mice neutralized heterologous pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2-RBD nanoparticle immunizations, demonstrating no loss of immunogenicity against particular RBDs resulting from co-display. A single immunization with mosaic RBD nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.
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http://dx.doi.org/10.1126/science.abf6840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928838PMC
February 2021

Single-Cell Sorting of HBsAg-Binding Memory B Cells from Human Peripheral Blood Mononuclear Cells and Antibody Cloning.

STAR Protoc 2020 Dec 16;1(3):100129. Epub 2020 Oct 16.

Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China.

The isolation of human antibodies with naturally paired heavy and light chains is crucial for understanding the human antibody immune response. Here, we present a protocol for antibody cloning from the sorted single human memory B cells recognizing hepatitis B virus (HBV) S antigen (HBsAg). A two-fluorescent-dye labeling strategy against HBsAg allows for an improved sorting specificity, while non-relevant protein staining allows for the exclusion of non-specific B cells. This protocol could also be widely adapted for other antigens. For complete details on the use and execution of this protocol, please refer to Wang et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2020.100129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757113PMC
December 2020

Sequence Evaluation and Comparative Analysis of Novel Assays for Intact Proviral HIV-1 DNA.

J Virol 2021 02 24;95(6). Epub 2021 Feb 24.

Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon antiretroviral therapy (ART)-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The intact proviral DNA assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR) is a lower-throughput assay that uses limiting dilution long-distance PCR amplification followed by quantitative PCR (qPCR) and near-full-length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another, but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long-distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size. The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART)-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.
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http://dx.doi.org/10.1128/JVI.01986-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8094944PMC
February 2021

Persistent Cellular Immunity to SARS-CoV-2 Infection.

bioRxiv 2020 Dec 9. Epub 2020 Dec 9.

Laboratory of Molecular Immunology, The Rockefeller University, New York, NY 10065, USA.

SARS-CoV-2 is responsible for an ongoing pandemic that affected millions of individuals around the globe. To gain further understanding of the immune response in recovered individuals we measured T cell responses in paired samples obtained an average of 1.3 and 6.1 months after infection from 41 individuals. The data indicate that recovered individuals show persistent polyfunctional SARS-CoV-2 antigen specific memory that could contribute to rapid recall responses. In addition, recovered individuals show enduring immune alterations in relative numbers of CD4 and CD8 T cells, expression of activation/exhaustion markers, and cell division.

Summary: We show that SARS-CoV-2 infection elicits broadly reactive and highly functional memory T cell responses that persist 6 months after infection. In addition, recovered individuals show enduring immune alterations in CD4 and CD8 T cells compartments.
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http://dx.doi.org/10.1101/2020.12.08.416636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743071PMC
December 2020

Neutralizing Activity of Broadly Neutralizing anti-HIV-1 Antibodies against Primary African Isolates.

J Virol 2020 Dec 9. Epub 2020 Dec 9.

Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA

Novel therapeutic and preventive strategies are needed to contain the HIV-1 epidemic. Broadly neutralizing human antibodies (bNAbs) with exceptional activity against HIV-1 are currently being tested in HIV-1 prevention trials. The selection of anti-HIV-1 bNAbs for clinical development was primarily guided by their neutralizing activity against HIV-1 Env pseudotyped viruses. Here we report on the neutralizing activity of 9 anti-HIV-1 bNAbs now in clinical development against 126 Clade A, C, D PBMC-derived primary African isolates. The neutralizing potency and breadth of the bNAbs tested was significantly reduced compared to pseudotyped viruses panels. The difference in sensitivity between pseudotyped viruses and primary isolates varied from 3- to nearly 100-fold depending on the bNAb and the HIV-1 clade. Thus, the neutralizing activity of bNAbs against primary African isolates differs from their activity against pseudovirus panels. The data have significant implications for interpreting the results of ongoing HIV-1 prevention trials. HIV remains a major public health problem worldwide, and new therapies and preventive strategies are necessary for controlling the epidemic. Broadly neutralizing antibodies (bNAbs) have been developed in the past decade to fill this gap. The neutralizing activity of these antibodies against diverse HIV strains has mostly been measured using Env-pseudotyped viruses, which overestimate bNAb coverage and potency. In this study we measured the neutralizing activity of nine bNAbs against clade A, C, and D HIV isolates derived from cells of African patients living with HIV and produced in peripheral blood mononuclear cells. We found that the coverage and potency of bNAbs were often significantly lower than what was predicted by Env-psuedotyped viruses, and that this decrease was related to the bNAb biding site class. This data is important for the planning and analysis of clinical trials that seek to evaluate bNAbs for the treatment and prevention of HIV infection in Africa.
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http://dx.doi.org/10.1128/JVI.01909-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092834PMC
December 2020