1. Elicitation and maturation of VRC01-class antibodies in transgenic mouse models, which allows a germline human IGHV1-2*02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. A strong VRC01-class predicted germline precursor binder, eOD-GT8 60mer, was able to elicit and enrich VRC01-class antibodies in this mouse model.

2. Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires with sequential immunization. The serum from the stepwise immunized mice exhibited cross-strain neutralizing activities and the mutation frequency of both the IGHV1-2*02 IgH and VRC01 IgL chains steadily increased.

 3. Glycan Masking Focuses Immune Responses to the HIV-1 CD4-Binding Site and Enhances Elicitation of VRC01-Class Precursor Antibodies. A substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. To mask irrelevant epitopes, we introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 and evaluated the mutants in a VRC01-class mouse model. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors.

4. Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization. The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) develop, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated that each of the five lineages was initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization.

5. Fusion Peptide Priming –Alone or in Cocktail with Env Trimer– Imprints Broad HIV-1-Neutralizing Responses with a Characteristic Early B Cell Signature. To optimize the immunization regimen and shorten the “neutralization-eclipse phase”, we analyzed plasma and antigen-specific B cells from 7 different immunization regimens in 32 macaques with a common boosting module comprising five FP/Trimer immunizations. We found that FP priming to imprint cross-reactive FP-directed HIV-neutralizing responses, with FP-trimer cocktail elicited the earliest cross-strain responses.

6. Fusion Peptide Priming Reduces Immune Responses to HIV-1 Envelope Trimer Base. Soluble ‘SOSIP’-stabilized envelope (Env) trimers are promising HIV-vaccine immunogens. However, they induce high titer responses against the glycan-free trimer base, which is occluded on native virions. To delineate the impact on base responses of priming with immunogens targeting the fusion peptide (FP) site of vulnerability, we quantified the prevalence of trimer-base antibody responses in 49 non-human primates (NHPs) immunized with various SOSIP-stabilized Env trimers and FP-carrier conjugates. We found that trimer-base responses accounted for ~90% of the overall trimer response in animals immunized with trimer only, ~70% in animals immunized with a cocktail of SOSIP-trimer and FP-conjugate, and ~30% in animals primed with FP-conjugate prior to trimer immunization, with neutralization breadth in FP-conjugate-primed animals correlated inversely with trimer-base responses.

The Respiratory Viruses Core (RVC) within the Molecular Immunoengineering Section (MIS) at the Vaccine Research Center is focused on the development and testing of countermeasures for respiratory diseases caused by RNA viruses. Activities in the RVC range from antigen design and preclinical immunogenicity testing to exploratory studies using samples from human clinical trials. We have a long-standing interest in respiratory syncytial virus (RSV), which can cause serious illness, particularly for infants and older adults, and have evaluated immune responses to the prefusion F subunit vaccine, DS-Cav1, in a phase I clinical trial. Clinical samples were assessed for neutralizing activity against RSV, and B cell and T cell responses to vaccination were also determined. We continue to explore adaptive immunity to RSV infection and vaccination in ongoing research projects.

Our work also includes a variety of resurging and emerging viral pathogens, including paramyxoviruses, Zika, SARS-CoV-2, and enterovirus D68 (EV-D68). This often involves assay and model development for proof-of-concept immunogenicity and protection studies. We perform a variety of serological assays and use tools such as multiparameter flow cytometry to evaluate cellular responses and for monoclonal antibody discovery. RVC efforts are closely integrated with that of the MIS, and often support VRC-wide vaccine development efforts.

The Molecular Immunoengineering Section (MIS) at the Vaccine Research Center aims to conceive novel vaccine concepts that elicit broad and potent protective immune responses against influenza virus and provide a mechanistic principle for designing vaccines for other hypervariable pathogens such as coronaviruses and HIV-1. MIS is dedicated to advancing vaccine immunogen design beyond structure-based protein engineering by combining concepts and principles from multiple disciplines, including, but not limited to, immunobiology, biochemistry, biophysics, nanotechnology, and computational biology. The mission of the MIS is to define the fundamental rules behind vaccine-elicited immunity. Our interests span from basic immunology and virology to translational sciences with the goal of advancing vaccine concepts that would radically improve immune responses to vaccines. Our work involves various biochemical, biophysical, structural, immunological, and computational techniques and tools. MIS efforts include development of “supraseasonal” and “pre-pandemic” influenza vaccine candidates, “mosaic” antigen display technology, high-throughput high-definition virus neutralization assay systems, and animal models that recapitulate key aspects of human responses to influenza, such as immunological imprinting, preexisting immunity, antibody specificity and repertoire, immunodominance, and pathogenesis. MIS is also integrated with the VRC’s Influenza Program by serving as the lead of the Vaccine Concepts team. The Influenza Program involves multiple VRC Sections and Programs with the goal of advancing candidate vaccines from bench to clinic.

My work at the NIH concentrates on two aspects of HIV infection: the control of HIV-infection by the immunologic mechanism and the description of the changes in the CD4 T cell  transcriptome caused by HIV-infection.  In collaboration with individuals at the VRC, the California Institute of Technology and the Ragon Institute I am responsible for a clinical trial in which an AAV vector carries the coding sequence for VRC07, a potent broadly neutralizing Ab, into muscle cells of HIV-infected individuals on effective anti-retroviral therapy. In some individuals production of VRC07 occurred at ug/ml serum quantities for over 3 years. Although this level of VRC07 is not protective, this study shows that it is possible to side-step some of the difficulties in producing an immunogen capable of inducing a broadly neutralizing antibody by using a viral vector to transduction muscle cells.  I have also established methods to identify and sort live HIV-infected CD4 T cells. Unlike matrix proteins, envelope proteins are fully mature when transported to the surface of CD4 T cells.  By using fluorescently labeled broadly neutralizing antibodies that bind HIV envelope protein expressed on the surface of CD4 T cells, it is possible to use index sorting to identify live HIV-infected CD4 T cells.  The transcriptomes of these cells are then characterized using RNA seq. We have used these methods to characterize the transcriptomes from HIV-infected peripheral CD4 T cells and in ACH2 cells transitioning from “latent-infection” to “active-infection”. These studies have allowed us to correlate markers of disease progression such as CD4 down regulation, viral RNA concentrations and viral RNA splice patterns, with activation of NF-B pathway and increased HIV-RNA transcription.  We are currently using these methods to identify and characterize the longitudinal effect of SHIV infection on the CD4 T cell transcriptome of individual SHIV infected CD4 T cells from rhesus macaque lymph nodes.