Andrew N. Macintyre

Background: Identification of immune correlates in murine gonorrhea models has been hampered by study-dependent differences in vaccine antigens and administration routes. We previously showed that detergent-detoxified outer membrane vesicles (dOMVs) isolated from a PorA-, PorB-, and RmpM-deficient meningococcal strain (ΔABR) elicit antibodies that cross-react with Neisseria gonorrhoeae and enhance gonococcal clearance in a mouse model of lower reproductive tract infection. In this study, we investigated whether (1) ΔABR dOMVs can protect mice from ascending gonococcal infection and (2) vaccination route influences immune responses. Methods: Mice were vaccinated subcutaneously (SC) or intraperitoneally (IP) and then vaginally inoculated with gonococci. Bioburden of mice was measured and assessed relative to ΔABR dOMV-induced cellular and humoral immune responses. Results: Subcutaneous and intraperitoneal vaccination accelerated gonococcal clearance from the lower and upper reproductive tract at similar rates. Probing of gonococcal protein microarrays with immune sera from the 2 groups identified multiple vaccine targets that were commonly immunogenic. Despite comparable clearance patterns in vaccinated mice, differences in immune induction were observed that were dependent on administration route. SC immunized mice demonstrated a neutrophil influx that correlated with decreased vaginal bioburden; higher serum bactericidal activity against nonsialylated gonococci was also noted. In contrast, IP immunization induced higher serum and vaginal IgA levels, serum bactericidal activity against sialylated gonococci, and antigonococcal opsonophagocytic killing activity of neutrophils. Conclusions: This work demonstrates that ΔABR dOMVs protect against ascending gonococcal infection and that cellular and functional antibody responses to the same candidate vaccine may vary depending on immunization route.

The critical importance of the thymus for generating new naive T cells that protect against novel infections and are tolerant to self-antigens has led to a recent revival of interest in monitoring thymic function in species other than humans and mice. Nonhuman primates such as rhesus macaques (Macaca mulatta) provide particularly useful animal models for translational research in immunology. In this study, we tested the performance of a 15-marker multicolor Ab panel for flow cytometric phenotyping of lymphocyte subsets directly from rhesus whole blood, with validation by thymectomy and T cell depletion. Immunohistochemical and multiplex RNA expression analysis of thymus tissue biopsies and molecular assays on PBMCs were used to further validate thymus function. Results identify Ab panels that can accurately classify rhesus naive T cells (CD3+CD45RA+CD197+ or CD3+CD28+CD95−) and recent thymic emigrants (CD8+CD28+CD95−CD103+CD197+) using just 100 µl of whole blood and commercially available fluorescent Abs. An immunohistochemical panel reactive with pan-cytokeratin (CK), CK14, CD3, Ki-67, CCL21, and TdT provides histologic evidence of thymopoiesis from formalin-fixed, paraffin-embedded thymus tissues. Identification of mRNAs characteristic of both functioning thymic epithelial cells and developing thymocytes and/or molecular detection of products of TCR gene rearrangement provide additional complementary methods to evaluate thymopoiesis, without requiring specific Abs. Combinations of multiparameter flow cytometry, immunohistochemistry, multiplex gene expression, and TCR excision circle assays can comprehensively evaluate thymus function in rhesus macaques while requiring only minimal amounts of peripheral blood or biopsied thymus tissue.

Current seasonal influenza virus vaccines induce responses primarily against immunodominant but highly plastic epitopes in the globular head of the hemagglutinin (HA) glycoprotein. Because of viral antigenic drift at these sites, vaccines need to be updated and readministered annually. To increase the breadth of influenza vaccine–mediated protection, we developed an antigenically complex mixture of recombinant HAs designed to redirect immune responses to more conserved domains of the protein. Vaccine-induced antibodies were disproportionally redistributed to the more conserved stalk of the HA without hindering, and in some cases improving, antibody responses against the head domain. These improved responses led to increased protection against homologous and heterologous viral challenges in both mice and ferrets compared with conventional vaccine approaches. Thus, antigenically complex protein mixtures can at least partially overcome HA head domain antigenic immunodominance and may represent a step toward a more universal influenza vaccine.

There is an urgent need for vaccines against Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea. Vaccination with an outer-membrane vesicle (OMV)-based Neisseria meningitidis (Nm) vaccine provides some protection from Ng; however, the mechanisms underlying this cross-protection are unknown. To address this need, we developed multiplexed bead-based assays for the relative quantification of human and mouse IgG and IgA against Ng antigens. The assays were evaluated for analyte independence, dilutional linearity, specificity, sensitivity, intra- and inter-assay variability, and robustness to sample storage conditions. The assay was then used to test samples from mice and humans immunized with an Nm-OMV vaccine.

Thrombocytopenia, hemorrhage, anemia, and infection are life-threatening issues following accidental or intentional radiation exposure. Since few therapeutics are available, safe and efficacious small molecules to mitigate radiation-induced injury need to be developed. Our previous study showed the synthetic TLR2/TLR6 ligand fibroblast stimulating lipopeptide (FSL-1) prolonged survival and provided MyD88-dependent mitigation of hematopoietic acute radiation syndrome (H-ARS) in mice. Although mice and humans differ in TLR number, expression, and function, nonhuman primate (NHP) TLRs are like those of humans; therefore, studying both animal models is critical for drug development. The objectives of this study were to determine the efficacy of FSL-1 on hematopoietic recovery in small and large animal models subjected to sublethal total body irradiation and investigate its mechanism of action. In mice, we demonstrate a lack of adverse effects, an easy route of delivery (subcutaneous) and efficacy in promoting hematopoietic progenitor cell proliferation by FSL-1. NHP given radiation, followed a day later with a single subcutaneous administration of FSL-1, displayed no adversity but showed elevated hematopoietic cells. Our analyses revealed that FSL-1 promoted red blood cell development and induced soluble effectors following radiation exposure. Cytologic analysis of bone marrow aspirates revealed a striking enhancement of mononuclear progenitor cells in FSL-1-treated NHP. Combining the efficacy of FSL-1 in promoting hematopoietic cell recovery with the lack of adverse effects induced by a single administration supports the application of FSL-1 as a viable countermeasure against H-ARS.

Nucleic acid vaccines have shown promising results in the clinic against infectious diseases and cancers. To robustly improve the vaccine efficacy and safety, we developed an approach to increase the intracellular stability of nucleic acids by transiently inhibiting lysosomal function in targeted tissues using sucrose. To achieve efficient and localized delivery of sucrose in animals, we designed a biomimetic lipid nanoparticle (LNP) to target the delivery of sucrose into mouse muscle cells. Using this approach, viral antigen expression in mouse muscle after DNA vaccination was substantially increased and prolonged without inducing local or systemic inflammation or toxicity. The same change in antigen expression would be achieved if the vaccine dose could be increased by 3,000 folds, which is experimentally and clinically impractical due to material restrictions and severe toxicity that will be induced by such a high dose of nucleic acids. The increase in antigen expression augmented the infiltration and activation of antigen-presenting cells, significantly improved vaccine-elicited humoral and T cell responses, and fully protected mice against the viral challenge at a low dose of vaccine. Based on these observations, we conclude that transient inhibition of lysosome function in target tissue by sucrose LNPs is a safe and potent approach to substantially improve nucleic acid-based vaccines.