Xueling Wu, MD, PhD

The overall research interest of Wu Lab is to study human B cell responses to viral infections by discovering and characterizing potent broadly neutralizing antibodies (bNAbs). The Wu lab has ongoing research in three areas:

1. HIV
2. Viruses with pandemic threats such as Influenza, Coronaviruses, and Ebolaviruses
3. Autoantibodies

HIV broadly neutralizing antibodies (bNAbs)

While at the Vaccine Research Center (VRC), NIAID, NIH, Dr. Wu contributed to the discovery of an important class of HIV bNAbs, VRC01 and its class that target the CD4-binding site of the viral envelope (Env) glycoprotein. VRC01 was later tested in the first “antibody mediated prevention” (AMP) human trial and demonstrated 75% efficacy of blocking VRC01-sensitive viral strains. The Wu Lab continues to optimize and apply advanced techniques such as 10X Genomics Immune Profiling to efficiently identify HIV bNAbs and mAbs that mediate antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).

Based on our surprise finding of two HIV bNAb lineages that class-switched to both IgG and IgA, published in Jia et al, Cell Host & Microbe 2020, 27: 963, we have for the first time identified bona fide HIV IgA bNAbs. Because HIV spreads mainly through mucosal exposures, the dominant IgA at mucosa compartments has long been the class of antibodies desired at the portal of entry to block infection. We have thus focused our bNAb efforts to IgA and aim to test whether IgA, in its mucosal form – secretory IgA (SIgA), is better than IgG at blocking infection in a SHIV macaque challenge model. Our research project aims to address the following questions:

• Compared to IgG, what is the frequency of IgA bNAb response to HIV infection?
• Compared to IgG, what are the epitopes that IgA bNAbs target on HIV Env?
• Compared to IgG, what are the sequence signatures of IgA bNAbs?
• Compared to IgG, would IgA, in its mucosal form SIgA, enhance its HIV neutralization potency in vitro?
• Compared to IgG, would IgA, in its mucosal form SIgA, better at blocking SHIV infection in a macaque challenge model?

Answers to these questions would help study design for more mucosal targeted antibody-mediated protections from HIV.

To facilitate HIV bNAb clinical trials, an accurate and sensitive bNAb susceptibility assay with a quick turnaround time is needed. The Wu Lab is actively working with other labs at ADARC/CUMC to develop an integrated approach with two prongs: a phenotypic (Env function) assay for bulk Env neutralization assessment with Env-barcoding and a genotypic (Env sequence) algorithm for high-throughput Env nanopore sequencing and prediction of bNAb susceptibility.

HIV env evolution and bNAb lineages

To understand how HIV bNAbs are developed and driven by HIV env gene evolution, the Wu Lab has longitudinally tracked env evolution and bNAb development in SHIV-infected rhesus macaques and HIV-infected humans. In Jia et al, J Virol 2016, 90: 4017, we reported 5 SHIV_SF162P3N-infected rhesus macaques and 9 HIV-infected humans who developed bNAbs. Of the 5 macaques, we performed longitudinal plasma neutralization and defined sequential development of NAbs in these animals. From three macaques, GB40, FF69, and FD83, we isolated sequential Env sequences and identified in each animal 3-5 sequential NAb waves from the initial narrow autologous to later broad responses. To gain insights to the NAb targets, we examined specific amino acid changes in Env over time and found that gp120 V5 was targeted by the 1st wave of NAbs, as reported in Jia et al, Viruses 2018, 10: 262. These findings led to our working model that over the course of viral infection, the initial autologous NAbs gradually broaden to gain neutralization breadth; under this model, we tested two possible mechanisms – “epitope drifting” and “epitope optimization” for NAb broadening in these animals. Our follow-up studies support the mechanism of “epitope optimization” for bNAb development. Of the 9 HIV-infected humans who developed bNAbs, we obtained longitudinal blood samples from 7 subjects and determined their bNAb dynamics. Like the SHIV-macaque study above, we are currently isolating sequential Envs from these human subjects to map Env targets for bNAbs and test if the mechanism of “epitope optimization” holds for bNAb development in these human subjects.

Derived from this project, a powerful tool that the Wu Lab has advanced is the B cell receptor (BCR) repertoire sequencing. We published the workflow of an unbiased 5’ RACE (rapid amplification of cDNA ends) PCR and Illumina MiSeq of BCR repertoires in Waltari et al, Frontiers in Immunology 2018, 9: 628. We generated the BCR repertoire sequence data from two SHIV-infected rhesus macaques. During the initial analysis of macaque BCR repertoires, we realized that the current macaque IGHV germline database is incomplete and thus obtained the IGHV germlines of 4 macaques included in our study. Our analyses revealed new macaque IGHV germline genes, thus contributing to the non-human primate IGHV germline gene collection and database. With the IGHV germlines determined for each macaque, we are applying the animal’s self-germline reference to analyze expressed BCR repertoires and antibody lineages.

HIV vaccine research

While at the Fred Hutchinson Cancer Research Center, Dr. Wu isolated a panel of mother-to-child transmitted Envs including BG505, which was later used for the first successful construction of soluble Env trimer protein (SOSIP) and extensively tested as an Env immunogen that closely resembles the HIV native Env trimer. Our isolated HIV bNAb M4008_N1 binds to a novel V3 crown epitope revealed in Chan et al, Nature Communications, 2021, 12: 6464, which provided the foundation for a V3-based Env immunogen study led by Dr. X-P Kong at NYU School of Medicine. The Wu Lab has been actively involved in and instrumental to Dr. Kong’s HIV vaccine programs, which recently yielded exciting results implicating a major breakthrough. The Wu Lab will remain committed to Dr. Kong’s efforts to develop a successful HIV vaccine.

Viruses with pandemic threats:

Since the global breakout of COVID-19 in 2020, NAbs and vaccines have been quickly identified and developed for treatment and prevention. However, viral escape from vaccines and NAbs has been a common theme for COVID-19. The next challenge and research interest for COVID-19 is to develop pan-coronavirus vaccines and identify truly broad and potent bNAbs, which may exist in a subset of convalescent cases and vaccinees. Working with Dr. Andres Finzi at University of Montreal, the Wu Lab has designed a probing strategy to identify broadly reactive B cells from a Canadian study cohort, and this work is currently ongoing. Since COVID-19 primarily targets the airway – a mucosal compartment, the Wu Lab is also investigating its IgA response, with an ongoing collaboration with Dr. Rebecca Powell at Mount Sinai to isolate mAbs from breastmilk of COVID-19 convalescent cases and vaccinees.

For Influenza, the Wu Lab has isolated four mAbs directed to the hemagglutinin (HA) from a 2013 H7N9 convalescent case in Hong Kong. Two HA1-directed mAbs neutralized H7N9 and protected mice from a lethal challenge. Cryo-EM structures revealed that these mAbs bind to a β14-centered surface partially overlapping with the antigenic site D of HA1 and disrupt the 220-loop that makes hydrophobic contacts with sialic acid on the adjacent protomer, thus affectively blocking viral entry. Both mAbs remained active to later H7N9 isolates from 2016-2017. The HA2-directed mAb lacked neutralizing activity but protected mice from lethal challenge when engineered to mouse IgG2a with Fc effector function. Used in combination with a suboptimal dose HA1-directed neutralizing mAb, the HA2-directed mAb augmented mouse protection. The Columbia University Tech Office has filed a provisional application to patent these mAbs. As Influenza is well known for its genetic reassortment and antigenic drift and shift, the next challenge and research interest for Influenza is also developing universal vaccines and finding truly broad and potent bNAbs, which is an interest of research in the Wu Lab. Since Influenza primarily targets the airway, the Wu Lab is also interested in its IgA response.

For Ebolaviruses, the current two in-use vaccines, rVSV-ZEBOV and a two-dose combination of Ad26.ZEBOV and MVA-BN-Filo, have yet to demonstrate protection against multiple Ebolavirus species. Our collaborators at the Southern Medical University in China immunized (I.M.) 6 Chinese rhesus macaques with Zaire, Sudan, and Bundibugyo glycoproteins (GPs) in DNA and protein to elicit bNAb responses. All immunized macaques mounted high plasma anti-GP and anti-Ebola neutralizing titers, demonstrating the feasibility for GP-based vaccines to elicit bNAbs that cover all three major Ebola species, Zaire, Sudan, and Bundibugyo. The top three macaques that developed the most robust cross-reactive NAbs can be used for bNAb isolation. However, mAbs have not been derived from these animals because the techniques for mAb isolation are not available on site at the Southern Medical University in China. With additional funding, the Wu Lab aims to ship these macaque specimens to ADARC/CUMC to isolate anti-Ebola bNAbs.

Autoantibodies

Recently during the COVID-19 pandemic, the Wu Lab has worked with Dr. Wei Jiang at Medical University of South Carolina to examine antibody responses following COVID-19 infection and mRNA vaccination. Based on our findings of elevated anti-IFN-α autoantibodies both in COVID-19 infection and mRNA vaccination cases, we proposed to further probe these autoantibodies for mechanisms of production, affinity maturation, antigen recognition, and immune regulatory functions. To isolate anti-IFN-α autoantibodies, the Wu Lab has developed a B cell probing strategy, i.e., using IFN-β, which has a relatively higher binding affinity to IFN α/β receptor (IFNAR) than IFN-α to block IFNAR on B cells, prior to staining BCRs with an IFN-α antigen. We have tested the staining and sorting strategy using PBMCs from a donor who presented heightened levels of plasma neutralizing anti-IFN-α autoantibodies. The sorted IFN-α+ B cells have been processed for 10X Genomics. We are in the process of characterizing mAbs from these sorted B cells and testing their functions to neutralize IFN-α-mediated activity. Although in acute viral infections such as COVID-19, anti-IFN-α autoantibodies are thought to dampen IFN-α-mediated antiviral activity thus fueling disease severity, these mAbs may be beneficial in treating inflammatory diseases and chronic viral infections where IFN-α plays a role in pathogenesis.

Dr. Wei Jiang also has a longstanding expertise in anti-CD4 autoantibodies in HIV-infected individuals. Up to 25% of people with HIV on antiretroviral therapy (ART) fail to restore peripheral CD4+ T cells to the levels of healthy controls. Dr. Jiang determined that anti-CD4 IgGs from non-responders (CD4+ T cell counts < 350 cells/μL) mediate CD4+ T cell death via ADCC and contribute to poor CD4+ T cell recovery from suppressive ART. The pathogenic ADCC anti-CD4 IgGs are even more profound in HIV-infected cocaine users than non-users. We have proposed to isolate anti-CD4 autoantibodies presenting ADCC activity, particularly in HIV-infected cocaine users, to further investigate the mechanisms of autoantibody production, affinity maturation, antigen recognition, and immune regulatory functions.

With well-established mAb expertise and techniques, the Wu Lab hopes to build an antibody-based biobankto zoonotic viruses with pandemic potential to prepare for future pandemics. After COVID-19, preparedness for future pandemics is an area of research with high enthusiasm. The viruses of interest may include additional coronaviruses such as bat RsSHC014-CoV, porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV), Influenza such as H1N1 G4 EA, as well as Marburg, Nipah, and Lassa viruses. We propose to immunize human antibody V-gene knock-in micethat are being developed by Genomab to affinity mature NAbs against the proposed zoonotic viruses, to which there is no or limited human antibody data available. Alternatively, Dr. Moriya Tsuji at ADARC/CUMC has developed human immune system (HIS) CD4+ T and B cell (CD4/B) mice that can be used for immunizations. Analysis of antiviral NAbs ahead of time offers critical knowledge of vulnerabilities on viral surface proteins that mediate viral entry, which is crucial to inform viral targets for vaccine development. The impact of this project, if successful, will be huge to provide potent NAbs as a quick response to future pandemics. With additional funding, the Wu Lab will be able to execute this idea of research.