Cassie Liu

Bio: Cassie Liu is an academic researcher from Rockefeller University. The author has contributed to research in topics: Antibody & Perforin. The author has an hindex of 9, co-authored 10 publications receiving 988 citations. Previous affiliations of Cassie Liu include Washington University in St. Louis & Howard Hughes Medical Institute.

Perforin and serine esterase gene expression in stimulated human T cells. Kinetics, mitogen requirements, and effects of cyclosporin A.

Abstract: A pore-forming protein (PFP; perforin) and various serine esterases (SE) have been identified in the cytoplasmic granules of CTL and NK cells. Perforin and several SE have recently been cloned. Northern blotting analysis was performed here using cDNA probes specific for human perforin and two SE (SE 1/HS and SE 2/GB) to monitor the levels of specific mRNAs in mitogen-stimulated primary human T cells. These mRNAs were rapidly induced by IL-2 with optimal responses at 300 U/ml. After IL-2 treatment, mRNAs for perforin, SE 1, and SE 2 peaked at 12-24 h and decreased after 48 h. The three mRNAs were also induced in T cells treated with a combination of PMA plus lectin, OKT3 mAb, or plastic-adherent accessory cells. However, the induction induced by PMA/mitogen followed a slower kinetics, peaking at 48 h. In general, we found that SE 1 mRNA was more readily induced by IL-2, while SE 2 responded better to PMA/mitogen. Similar patterns of mRNA expression were observed for both unprimed T cells and PHA-primed T blasts. After stimulation with IL-2 and PMA/mitogen, the T8+ subset was shown to be the main producer of perforin, SE 1, and SE 2. Low levels of all three mRNAs, however, were also detected in the T4+ subset. The induction of all three mRNAs by either IL-2 or PMA/mitogen was partially blocked by the immunosuppressive drug cyclosporin A (CsA), but not by the biologically inactive analogue cyclosporin H. Together, these results point to some similarities and differences with upregulation of granule mediator mRNAs relative to lymphokine mRNAs. Both sets of genes require two signals for their induction by mitogens. In contrast to lymphokines, there is a strong response of granule mRNAs to IL-2, and the induction of these transcripts is only partially blocked by CsA.

Sequencing and cloning of antigen-specific antibodies from mouse memory B cells

Abstract: Methods to identify genes encoding immunoglobulin heavy and light chains from single B lymphocytes vary in efficiency, error rate and practicability. Here we describe a protocol to sequence and clone the variable antibody region of single antigen-specific mouse memory B cells for antibody production. After purification, antigen-specific mouse memory B cells are first single-cell-sorted by fluorescence-activated cell sorting (FACS), and V(D)J transcripts are amplified by RT-PCR. Fragments are then combined with linearized expression vectors, assembled in vitro as part of a sequence- and ligation-independent cloning (SLIC) reaction and then transformed into Escherichia coli. Purified vectors can then be used to produce monoclonal antibodies in HEK293E suspension cells. This protocol improves the amplification efficiency of antibody variable genes and accelerates the cloning workflow. Antibody sequences will be available in 3-4 d, and microgram to milligram amounts of antibodies are produced within 14 d. The new protocol should be useful for addressing fundamental questions about antigen-specific memory B cell responses, as well as for characterizing antigen-specific antibodies.

Improving Neutralization Potency and Breadth by Combining Broadly Reactive HIV-1 Antibodies Targeting Major Neutralization Epitopes

Abstract: The isolation of broadly neutralizing HIV-1 monoclonal antibodies (MAbs) to distinct epitopes on the viral envelope glycoprotein (Env) provides the potential to use combinations of MAbs for prevention and treatment of HIV-1 infection. Since many of these MAbs have been isolated in the last few years, the potency and breadth of MAb combinations have not been well characterized. In two parallel experiments, we examined the in vitro neutralizing activities of double-, triple-, and quadruple-MAb combinations targeting four distinct epitopes, including the CD4-binding site, the V1V2-glycan region, the V3-glycan supersite, and the gp41 membrane-proximal external region (MPER), using a panel of 125 Env-pseudotyped viruses. All MAb combinations showed substantially improved neutralization breadth compared to the corresponding single MAbs, while the neutralization potency of individual MAbs was maintained. At a 50% inhibitory concentration (IC 50 ) cutoff of 1 μg/ml per antibody, double-MAb combinations neutralized 89 to 98% of viruses, and triple combinations neutralized 98 to 100%. Overall, the improvement of neutralization breadth was closely predicted by an additive-effect model and explained by complementary neutralization profiles of antibodies recognizing distinct epitopes. Subtle but consistent favorable interactions were observed in some MAb combinations, whereas less favorable interactions were observed on a small subset of viruses that are highly sensitive to V3-glycan MAbs. These data demonstrate favorable in vitro combinations of broadly neutralizing HIV-1 MAbs and suggest that such combinations could have utility for HIV-1 prevention and treatment. IMPORTANCE Over the last 5 years, numerous broadly reactive HIV-1-neutralizing MAbs have been isolated from B cells of HIV-1-infected donors. Each of these MAbs binds to one of the major vulnerable sites (epitopes) on the surface of the viral envelope glycoprotein. Since antibodies to distinct viral epitopes could theoretically act together to provide greater potency and breadth of virus neutralization, we tested physical mixtures of double, triple, and quadruple combinations of neutralizing MAbs targeting four major epitopes on HIV-1 Env. When tested together, antibody combinations showed substantially improved neutralization breadth compared to single MAbs. This improvement could be explained by the complementary neutralization profiles of individual MAbs. We further demonstrated that each antibody maintained its full neutralization potency when used in combination with other MAbs. These data provide a rationale for clinical use of antibody-based combinations for HIV-1 prevention and therapy.

Convergent antibody responses to SARS-CoV-2 in convalescent individuals.

Abstract: During the COVID-19 pandemic, SARS-CoV-2 infected millions of people and claimed hundreds of thousands of lives Virus entry into cells depends on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein (S) Although there is no vaccine, it is likely that antibodies will be essential for protection However, little is known about the human antibody response to SARS-CoV-21-5 Here we report on 149 COVID-19 convalescent individuals Plasmas collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres: less than 1:50 in 33% and below 1:1,000 in 79%, while only 1% showed titres above 1:5,000 Antibody sequencing revealed expanded clones of RBD-specific memory B cells expressing closely related antibodies in different individuals Despite low plasma titres, antibodies to three distinct epitopes on RBD neutralized at half-maximal inhibitory concentrations (IC50 values) as low as single digit nanograms per millitre Thus, most convalescent plasmas obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective

Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117

Abstract: HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg^(−1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8–2.5 log_(10) and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.

Death by a thousand cuts: granzyme pathways of programmed cell death.

Abstract: The granzymes are cell death–inducing enzymes, stored in the cytotoxic granules of cytotoxic T lymphocytes and natural killer cells, that are released during granule exocytosis when a specific virus-infected or transformed target cell is marked for elimination. Recent work suggests that this homologous family of serine esterases can activate at least three distinct pathways of cell death. This redundancy likely evolved to provide protection against pathogens and tumors with diverse strategies for evading cell death. This review discusses what is known about granzyme-mediated pathways of cell death as well as recent studies that implicate granzymes in immune regulation and extracellular proteolytic functions in inflammation.

Antiretroviral Drugs for Treatment and Prevention of HIV Infection in Adults: 2018 Recommendations of the International Antiviral Society–USA Panel

Abstract: Importance Antiretroviral therapy (ART) is the cornerstone of prevention and management of HIV infection. Objective To evaluate new data and treatments and incorporate this information into updated recommendations for initiating therapy, monitoring individuals starting therapy, changing regimens, and preventing HIV infection for individuals at risk. Evidence Review New evidence collected since the International Antiviral Society–USA 2016 recommendations via monthly PubMed and EMBASE literature searches up to April 2018; data presented at peer-reviewed scientific conferences. A volunteer panel of experts in HIV research and patient care considered these data and updated previous recommendations. Findings ART is recommended for virtually all HIV-infected individuals, as soon as possible after HIV diagnosis. Immediate initiation (eg, rapid start), if clinically appropriate, requires adequate staffing, specialized services, and careful selection of medical therapy. An integrase strand transfer inhibitor (InSTI) plus 2 nucleoside reverse transcriptase inhibitors (NRTIs) is generally recommended for initial therapy, with unique patient circumstances (eg, concomitant diseases and conditions, potential for pregnancy, cost) guiding the treatment choice. CD4 cell count, HIV RNA level, genotype, and other laboratory tests for general health and co-infections are recommended at specified points before and during ART. If a regimen switch is indicated, treatment history, tolerability, adherence, and drug resistance history should first be assessed; 2 or 3 active drugs are recommended for a new regimen. HIV testing is recommended at least once for anyone who has ever been sexually active and more often for individuals at ongoing risk for infection. Preexposure prophylaxis with tenofovir disoproxil fumarate/emtricitabine and appropriate monitoring is recommended for individuals at risk for HIV. Conclusions and Relevance Advances in HIV prevention and treatment with antiretroviral drugs continue to improve clinical management and outcomes for individuals at risk for and living with HIV.