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.

We apply systems immunology approaches to investigate human cutaneous leishmaniasis, a disfiguring chronic skin disease caused by the protozoan parasite Leishmania.

Cutaneous leishmaniasis (CL) is endemic and emerging in the southwestern United States and often also affects military personnel who have been deployed to Leishmania-endemic areas. Leishmania parasites are transmitted to humans via the bite of an infected sand fly. As the disease progresses, the immune response to the parasite in the skin can trigger pathologic inflammation that leads to chronic ulceration and permanent scarring. Current therapies for CL are variably effective with increasing reports of drug resistance. A major roadblock to the development of novel therapeutics for CL is our incomplete understanding of human immunity to Leishmania in the skin.

Our group seeks to define the cellular circuits that drive pathologic inflammation and damage to skin tissue in CL. To do this, we employ a variety of experimental and bioinformatic approaches, including single-cell transcriptomics, multiomic spatial profiling, and mechanistic multiscale tissue modeling to map the cell subsets and intercellular signals that drive CL immunopathology. Current efforts are focused on constructing a comprehensive single-cell and spatial atlas of human CL. This work is being performed in collaboration with the NIAID Leishmaniasis Clinic (Principal Investigator: Dr. Elise O’Connell, Laboratory of Parasitic Diseases). Our goal is to develop novel host-directed therapies for CL patients that 1) inhibit the critical signaling circuits driving pathologic inflammation in the skin and 2) enhance immune control of the parasite.

Another major focus of our group is investigating human immunity to vector bites and vector-borne pathogens via controlled human vector challenge studies. The immune response to vector bites profoundly affects the susceptibility of the host to vector-borne pathogens. In collaboration with Dr. Matthew Memoli (Laboratory of Infectious Diseases), we performed a human challenge study using mosquitos and sand flies, which revealed a conserved transcriptomic skin response against highly divergent vector species. We are now using single-cell and spatial profiling to map the cellular circuitry of the human skin response to vector bites. Our goal is to develop novel clinical therapies that co-opt these responses to enhance protection against vector-borne pathogens.

Dr. Carroll’s primary research is concerned with understanding how neuroinflammation and glial cell activation (astrocytes and microglia) influence prion pathogenesis and neurodegeneration. Prions are infectious misfolded conformers of the cellular GPI-anchored protein PrP and can spread from cell to cell within the brain by seeded-polymerization. This group of proteinopathies can affect humans, cattle, sheep, and cervids and are resistant to many standard decontamination methods. Initially, it was assumed that prion diseases lack an immunological component due to the absence of a prominent antibody or interferon response. However, Dr. Carroll’s research has shown that prion infection elicits a substantial inflammatory response in the CNS and that many inflammatory effectors increase in expression in response to prion infection.

To address the potential impact of microglia in prion disease, Dr. Carroll performed several studies using the potent CSF-R1 inhibitor PLX5622 to reduce microglia in the CNS. These studies indicated that microglia were indispensable to host defense against prion disease. Moreover, his research implicated astrocytes as potentially affecting pathology during disease, where when microglia were absent, the astrocytes were more highly active, expressing numerous disease-related components. This has led to further investigations to assess astrogliosis during prion infection.

A Uniform Manifold Approximation and Projection (UMAP) of single nuclei RNA (snRNA) sequencing analysis of uninfected and prion-infected mouse thalamus depicting 43 transcriptional clusters from >69,000 nuclei examined.

Credit: NIAID

Using high-throughput deep sequencing of RNA transcripts in longitudinal studies, Dr. Carroll has identified numerous differentially expressed genes in the CNS during prion infection. These investigations have yielded compelling results and suggest that microglia in the prion-infected brain assume an alternative phenotype that is distinct from those seen in other brain disorders. From these RNA-seq studies, it was determined that reactive astrocytes assume an expression signature that is not reliant on the canonical signals described in other neuroinflammatory models. Furthermore, this prion-specific reactive astrocyte expression signature is exacerbated when microglia are reduced in the CNS.

Dr. Carroll has begun to analyze the individual cellular changes in the brain using single nuclei RNA (snRNA) sequencing to better understand the relevant changes in the cell populations in the complex milieu of the CNS during infection. Thus far, the research has focused on gene expression changes in the thalamus at pre- and early-clinical times. The thalamus is affected early during prion infection in rodent models, and thalamic pathology is a key feature in human forms of prion disease.

A new aspect of Dr. Carroll’s research is a collaboration with Dr. Cathryn Haigh (Chief, Prion Cell Biology Unit, NIAID) to study Lyme Neuroborreliosis (LNB). Lyme disease, a global public health concern, is the most common tick-borne disease in North America and Eurasia, with an estimated 14% of the world’s population having become infected. Reported cases of Lyme disease in the U.S. have been on the rise for many years, with over 62,000 confirmed cases reported in 2022, making it the leading reportable arthropod-borne infectious disease. The Centers for Disease Control estimates that the disease is underreported, and the true incidence of Lyme disease in the U.S. is approaching 500,000 cases annually. Lyme disease, caused by bacterial spirochetes of the genus Borrelia, is a multi-systemic disorder affecting the skin, heart, central nervous system, and joints.

To address the need for additional models of LNB and to better understand the responses of the human CNS when exposed to Borrelia, this collaboration has developed two in vitro model systems. The first uses human cerebral organoids differentiated from human induced pluripotent stem cells (iPSCs) as an in vitro tissue model. The second uses iPSCs for differentiation into specific neuronal subtypes, astrocytes, and microglia-like cells for study. Exploiting these human-derived model systems, we are assessing responses to Borrelia infection that are stimulated in isolated cells from specific responses that only occur in these cells when they are part of an integrated organoid network. This project incorporates several cutting-edge technologies, including organoid development, bulk and single-cell RNA sequencing, metabolomics, and lipidomics.

Experimental design and strategy to address potential responses of human cerebral organoids, astrocytes, and neurons after exposure to infectious Borrelia species that cause Lyme Neuroborreliosis.

Credit: NIAID

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.

We are currently investigating the role of mutant p53-induced NOX4 on the cancer cell secretome, and the effects NOX4-derived reactive oxygen species have on the inflammatory tumor microenvironment.

Dr. De Ravin’s primary goal is to develop novel gene therapy and cell therapy approaches for treatment of Inborn Errors of Immunity (IEI) /Primary Immunodeficiency Diseases (PID). Advances in genomic diagnosis and immune-phenotype characterization within National Institutes of Allergy and Infectious Diseases identifies many individuals who will benefit from gene and cell therapy.  Current gene therapy for IEI (e.g., X-linked SCID, Chronic Granulomatous Disease (CGD) using lentivectors) has provided clinical benefit to multiple patients. However, the risk semi-random vector insertion causing insertional oncogenesis and the lack of physiological gene expression from inserted exogenous transgenes leave room for improvement. To address these concerns, Dr. De Ravin is working on targeted approaches to insert therapeutic genes in hematopoietic stem and progenitor cells for future gene therapy.  Programmable CRISPR-Cas9 nuclease systems can deliver corrective genes or repair mutations efficiently. However, this approach carries risks of genotoxicity although there are mitigating agents. Base editing that side-steps risks associated with double strand DNA breaks and the dependence on homology-directed repair is another promising approach for gene therapy for the near future. For patients with infections difficult to control with current antimicrobials, mRNA transfection of autologous primary cells such as granulocytes provide hopes for a transient therapeutic approach. A better understanding of the immune-phenotype allows rational designs for short-term disease control and ultimately long-term treatment of disease with ideal gene therapy approach.

Inborn Errors of Immunity (IEIs) are genetic disorders of the immune system with clinical manifestations of infection and autoinflammatory syndrome. Neurological disorders are one of the common causes of irreversible morbidity and mortality in patients with IEIs. Neuroinflammatory, neuroinfectious and neurodegenerative diseases have been reported extensively in this patient population but the role of immune related gene defects on development, function and immunoregulation of nervous system is still unknown.

The NeuroImmunopathogenesis Unit performs an integrated bench to bedside research to better understand the role of immunodeficiencies in nervous system. Taking a comprehensive approach to evaluate profile and function of immune cells in both blood and cerebrospinal fluid, the most adjacent cells to nervous system, provides valuable understanding about dynamic of immune cells and responses in CNS immune-compartment paving the path to find more targeted therapeutics. By using induced pluripotent stem cell technology, the unit also investigates the role of immune related gene defects in development and function of human neurons and glia to find underlying cellular and molecular pathways in immunodeficient patients with neurological disorders.

Furthermore, rare neuroinfectious, neuroinflammatory and neurodegenerative diseases with atypical clinical features can be a manifestation of immune related gene defects. Our holistic clinical and basic immunology, neuroscience and genetic approach facilitates to better understand the underlying mechanisms of these presentations to clarify diagnosis and treatments of this patients’ complex and often refractory to treatment neurological diseases.

The overall goal of Dr. Manthiram’s research is to elucidate genetic susceptibility factors and immunologic mechanisms of mucosal autoinflammatory disorders through translational research. 

Her research group studies periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome, which is the most common periodic fever syndrome in children. Patients with PFAPA have recurrent, regular episodes of fever with aphthous ulcers pharyngitis, and cervical adenitis.  She has focused on unraveling the pathogenesis of PFAPA syndrome by identifying genetic risk factors and studying tonsil immunology. She has identified common genetic susceptibility loci and class I and class II HLA risk alleles for PFAPA indicating the PFAPA is a complex genetic disease. These associated risk variants are also risk alleles for recurrent aphthous ulcers and Behçet’s disease, which links these three oropharyngeal disorders on a spectrum of disease called Behçet’s spectrum disorders. Recently, her group has studied the immune response to SARS-CoV-2 in the tonsil and adenoid tissue, shedding light on the mucosal immune response to this virus.

Her lab also studies the mechanism of other mucosal inflammatory disorders including obstructive sleep apnea and trisomy 8-associated autoinflammatory disease (TRIAD). Dr. Manthiram follows patients with PFAPA and TRIAD at NIH.