Lilit Grigoryan

All Publications

• Phosphorylcholine-conjugated gold-molecular clusters improve signal for Lymph Node NIR-II fluorescence imaging in preclinical cancer models. Nature communications Baghdasaryan, A., Wang, F., Ren, F., Ma, Z., Li, J., Zhou, X., Grigoryan, L., Xu, C., Dai, H. 2022; 13 (1): 5613

  Abstract

  Sentinel lymph node imaging and biopsy is important to clinical assessment of cancer metastasis, and novel non-radioactive lymphographic tracers have been actively pursued over the years. Here, we develop gold molecular clusters (Au25) functionalized by phosphorylcholine (PC) ligands for NIR-II (1000-3000nm) fluorescence imaging of draining lymph nodes in 4T1 murine breast cancer and CT26 colon cancer tumor mouse models. The Au-phosphorylcholine (Au-PC) probes exhibit 'super-stealth' behavior with little interactions with serum proteins, cells and tissues in vivo, which differs from the indocyanine green (ICG) dye. Subcutaneous injection of Au-PC allows lymph node mapping by NIR-II fluorescence imaging at an optimal time of ~ 0.5 - 1hour postinjection followed by rapid renal clearance. Preclinical NIR-II fluorescence LN imaging with Au-PC affords high signal to background ratios and high safety and biocompatibility, promising for future clinical translation.

  View details for DOI 10.1038/s41467-022-33341-6

  View details for PubMedID 36153336

• Adjuvanting a subunit SARS-CoV-2 vaccine with clinically relevant adjuvants induces durable protection in mice. NPJ vaccines Grigoryan, L., Lee, A., Walls, A. C., Lai, L., Franco, B., Arunachalam, P. S., Feng, Y., Luo, W., Vanderheiden, A., Floyd, K., Wrenn, S., Pettie, D., Miranda, M. C., Kepl, E., Ravichandran, R., Sydeman, C., Brunette, N., Murphy, M., Fiala, B., Carter, L., Coffman, R. L., Novack, D., Kleanthous, H., O'Hagan, D. T., van der Most, R., McLellan, J. S., Suthar, M., Veesler, D., King, N. P., Pulendran, B. 2022; 7 (1): 55

  Abstract

  Adjuvants enhance the magnitude and the durability of the immune response to vaccines. However, there is a paucity of comparative studies on the nature of the immune responses stimulated by leading adjuvant candidates. In this study, we compared five clinically relevant adjuvants in mice-alum, AS03 (a squalene-based adjuvant supplemented with α-tocopherol), AS37 (a TLR7 ligand emulsified in alum), CpG1018 (a TLR9 ligand emulsified in alum), O/W 1849101 (a squalene-based adjuvant)-for their capacity to stimulate immune responses when combined with a subunit vaccine under clinical development. We found that all four of the adjuvant candidates surpassed alum with respect to their capacity to induce enhanced and durable antigen-specific antibody responses. The TLR-agonist-based adjuvants CpG1018 (TLR9) and AS37 (TLR7) induced Th1-skewed CD4+ T cell responses, while alum, O/W, and AS03 induced a balanced Th1/Th2 response. Consistent with this, adjuvants induced distinct patterns of early innate responses. Finally, vaccines adjuvanted with AS03, AS37, and CpG1018/alum-induced durable neutralizing-antibody responses and significant protection against the B.1.351 variant 7 months following immunization. These results, together with our recent results from an identical study in non-human primates (NHPs), provide a comparative benchmarking of five clinically relevant vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV-2 subunit vaccines to provide durable protection against the B.1.351 variant. Furthermore, these results reveal differences between the widely-used C57BL/6 mouse strain and NHP animal models, highlighting the importance of species selection for future vaccine and adjuvant studies.

  View details for DOI 10.1038/s41541-022-00472-2

  View details for PubMedID 35606518

• Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine. Nature immunology Li, C., Lee, A., Grigoryan, L., Arunachalam, P. S., Scott, M. K., Trisal, M., Wimmers, F., Sanyal, M., Weidenbacher, P. A., Feng, Y., Adamska, J. Z., Valore, E., Wang, Y., Verma, R., Reis, N., Dunham, D., O'Hara, R., Park, H., Luo, W., Gitlin, A. D., Kim, P., Khatri, P., Nadeau, K. C., Pulendran, B. 2022

  Abstract

  Despite the success of the BNT162b2 mRNA vaccine, the immunological mechanisms that underlie its efficacy are poorly understood. Here we analyzed the innate and adaptive responses to BNT162b2 in mice, and show that immunization stimulated potent antibody and antigen-specific T cell responses, as well as strikingly enhanced innate responses after secondary immunization, which was concurrent with enhanced serum interferon (IFN)-gamma levels 1d following secondary immunization. Notably, we found that natural killer cells and CD8+ T cells in the draining lymph nodes are the major producers of this circulating IFN-gamma. Analysis of knockout mice revealed that induction of antibody and T cell responses to BNT162b2 was not dependent on signaling via Toll-like receptors 2, 3, 4, 5 and 7 nor inflammasome activation, nor the necroptosis or pyroptosis cell death pathways. Rather, the CD8+ T cell response induced by BNT162b2 was dependent on type I interferon-dependent MDA5 signaling. These results provide insights into the molecular mechanisms by which the BNT162b2 vaccine stimulates immune responses.

  View details for DOI 10.1038/s41590-022-01163-9

  View details for PubMedID 35288714

• Systems biological assessment of human immunity to BNT162b2 mRNA vaccination. Research square Arunachalam, P. S., Scott, M. K., Hagan, T., Li, C., Feng, Y., Wimmers, F., Grigoryan, L., Trisal, M., Edara, V. V., Lai, L., Chang, S. E., Feng, A., Dhingra, S., Shah, M., Lee, A. S., Chinthrajah, S., Sindher, T., Mallajosyula, V., Gao, F., Sigal, N., Kowli, S., Gupta, S., Pellegrini, K., Tharp, G., Maysel-Auslender, S., Bosinger, S., Maecker, H. T., Boyd, S. D., Davis, M. M., Utz, P. J., Suthar, M. S., Khatri, P., Nadeau, K. C., Pulendran, B. 2021

  Abstract

  The emergency use authorization of two COVID-19 mRNA vaccines in less than a year since the emergence of SARS-CoV-2, represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems biological approach to comprehensively profile the innate and adaptive immune responses in 56 healthy volunteers vaccinated with the Pfizer-BioNTech mRNA vaccine. Vaccination resulted in robust production of neutralizing antibodies (nAbs) against the parent strain and the variant of concern, B.1.351, but no induction of autoantibodies, and significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. The innate response induced within the first 2 days of booster vaccination was profoundly increased, relative to the response at corresponding times after priming. Thus, there was a striking increase in the: (i) frequency of CD14+CD16+ inflammatory monocytes; (ii) concentration of IFN- y in the plasma, which correlated with enhanced pSTAT3 and pSTAT1 levels in monocytes and T cells; and (iii) transcriptional signatures of innate responses characteristic of antiviral vaccine responses against pandemic influenza, HIV and Ebola, within 2 days following booster vaccination compared to primary vaccination. Consistent with these observations, single-cell transcriptomics analysis of 242,479 leukocytes demonstrated a ~100-fold increase in the frequency of a myeloid cluster, enriched in a signature of interferon-response transcription factors (TFs) and reduced in AP-1 TFs, one day after secondary immunization, at day 21. Finally, we delineated distinct molecular pathways of innate activation that correlate with CD8 T cell and nAb responses and identified an early monocyte-related signature that was associated with the breadth of the nAb response against the B1.351 variant strain. Collectively, these data provide insights into the immune responses induced by mRNA vaccines and demonstrate their capacity to stimulate an enhanced innate response following booster immunization.

  View details for DOI 10.21203/rs.3.rs-438662/v1

  View details for PubMedID 34013244

  View details for PubMedCentralID PMC8132234

• Systems vaccinology of the BNT162b2 mRNA vaccine in humans. Nature Arunachalam, P. S., Scott, M. K., Hagan, T., Li, C., Feng, Y., Wimmers, F., Grigoryan, L., Trisal, M., Edara, V. V., Lai, L., Chang, S. E., Feng, A., Dhingra, S., Shah, M., Lee, A. S., Chinthrajah, S., Sindher, S. B., Mallajosyula, V., Gao, F., Sigal, N., Kowli, S., Gupta, S., Pellegrini, K., Tharp, G., Maysel-Auslender, S., Hamilton, S., Aoued, H., Hrusovsky, K., Roskey, M., Bosinger, S. E., Maecker, H. T., Boyd, S. D., Davis, M. M., Utz, P. J., Suthar, M. S., Khatri, P., Nadeau, K. C., Pulendran, B. 2021

  Abstract

  The emergency use authorization of two mRNA vaccines in less than a year since the emergence of SARS-CoV-2 represents a landmark in vaccinology1,2. Yet, how mRNA vaccines stimulate the immune system to elicit protective immune responses is unknown. Here we used a systems vaccinology approach to comprehensively profile the innate and adaptive immune responses of 56 healthy volunteers vaccinated with the Pfizer-BioNTech mRNA vaccine. Vaccination resulted in robust production of neutralizing antibodies (nAbs) against the parent Wuhan strain and, to a lesser extent, the B.1.351 strain, and significant increases in antigen-specific polyfunctional CD4 and CD8 T cells after the second dose. Booster vaccination stimulated a strikingly enhanced innate immune response compared to primary vaccination, evidenced by a greater: (i) frequency of CD14+CD16+ inflammatory monocytes; (ii) concentration of plasma IFN-g; (iii) transcriptional signature of innate antiviral immunity. Consistent with these observations, single-cell transcriptomics analysis demonstrated a ~100-fold increase in the frequency of a myeloid cell cluster, enriched in interferon-response transcription factors (TFs) and reduced in AP-1 TFs, following secondary immunization. Finally, we identified distinct innate pathways associated with CD8 T cell and nAb responses, and show that a monocyte-related signature correlates with the nAb response against the B.1.351 variant strain. Collectively, these data provide insights into immune responses induced by mRNA vaccination and demonstrate its capacity to prime the innate immune system to mount a more potent response following booster immunization.

  View details for DOI 10.1038/s41586-021-03791-x

  View details for PubMedID 34252919

• The immunology of SARS-CoV-2 infections and vaccines. Seminars in immunology Grigoryan, L., Pulendran, B. 2020: 101422

  Abstract

  SARS-CoV-2, the virus that causes COVID-19, emerged in late 2019, and was declared a global pandemic on March 11th 2020. With over 50 million cases and 1.2 million deaths around the world, to date, this pandemic represents the gravest global health crisis of our times. Thus, the race to develop a COVID-19 vaccine is an urgent global imperative. At the time of writing, there are over 165 vaccine candidates being developed, with 33 in various stages of clinical testing. In this review, we discuss emerging insights about the human immune response to SARS-CoV-2, and their implications for vaccine design. We then review emerging knowledge of the immunogenicity of the numerous vaccine candidates that are currently being tested in the clinic and discuss the range of immune defense mechanisms that can be harnessed to develop novel vaccines that confer durable protection against SARS-CoV-2. Finally, we conclude with a discussion of the potential role of a systems vaccinology approach in accelerating the clinical testing of vaccines, to meet the urgent needs posed by the pandemic.

  View details for DOI 10.1016/j.smim.2020.101422

  View details for PubMedID 33262067

• Squalene-based adjuvants stimulate CD8 T cell, but not antibody responses, through a RIPK3-dependent pathway. eLife Kim, E. H., Woodruff, M. C., Grigoryan, L., Maier, B., Lee, S. H., Mandal, P., Cortese, M., Natrajan, M. S., Ravindran, R., Ma, H., Merad, M., Gitlin, A. D., Mocarski, E. S., Jacob, J., Pulendran, B. 2020; 9

  Abstract

  The squalene-based oil-in-water emulsion (SE) vaccine adjuvant MF59 has been administered to more than 100 million people in more than 30 countries, in both seasonal and pandemic influenza vaccines. Despite its wide use and efficacy, its mechanisms of action remains unclear. In this study we demonstrate that immunization of mice with MF59 or its mimetic AddaVax (AV) plus soluble antigen results in robust antigen-specific antibody and CD8 T cell responses in lymph nodes and non-lymphoid tissues. Immunization triggered rapid RIPK3-kinase dependent necroptosis in the lymph node which peaked at 6 hours, followed by a sequential wave of apoptosis. Immunization with alum plus antigen did not induce RIPK3 kinase-dependent signaling. RIPK3-dependent signaling induced by MF59 or AV was essential for cross-presentation of antigen to CD8 T cells by Batf3-dependent CD8+ DCs. Consistent with this, RIPK3-kinase deficient or Batf3 deficient mice were impaired in their ability to mount adjuvant-enhanced CD8 T cell responses. However, CD8 T cell responses were unaffected in mice deficient in MLKL, a downstream mediator of necroptosis. Surprisingly, antibody responses were unaffected in RIPK3-kinase or Batf3 deficient mice. In contrast, antibody responses were impaired by in vivo administration of the pan-caspase inhibitor Z-VAD-FMK, but normal in caspase-1 deficient mice, suggesting a contribution from apoptotic caspases, in the induction of antibody responses. These results demonstrate that squalene-based vaccine adjuvants induce antigen-specific CD8 T cell and antibody responses, through RIPK3-dependent and-independent pathways, respectively.

  View details for DOI 10.7554/eLife.52687

  View details for PubMedID 32515732

• Lacrimal Gland NK Cells Are Developmentally and Functionally Similar to Conventional NK Cells. ImmunoHorizons Erick, T., Grigoryan, L., Brossay, L. 2017; 1 (2): 2-9

  Abstract

  The murine lacrimal gland (LG), which produces crucial components of the ocular tear film, contains a population of natural killer (NK) cells. LG NK cells appear to belong to the conventional NK cell lineage, based on their cell surface receptor and transcription factor expression, absence in NFIL3-/- mice, and lack of RORγt expression during development. LG NK cells produce IFN-γ during the early stages of systemic murine cytomegalovirus (MCMV) infection. This effector response occurs in the absence of noticeable MCMV replication in the LG, indicating that LG NK cells are being activated by soluble factors. However, the magnitude of LG NK cell IFN-γ production during MCMV infection is significantly lower than spleen and liver NK cells. Adoptive transfer experiments in lymphopenic mice revealed that this hyporesponsive phenotype is tissue-specific, which indicates that that LG NK cells can produce a robust effector response.

  View details for DOI 10.4049/immunohorizons.1700008

  View details for PubMedID 28966997

  View details for PubMedCentralID PMC5616209