Dendritic cells (DC) are required for the immune response against Listeria monocytogenes and are permissive for infection in vivo and in vitro. However, it is unclear if DC provide a desirable intracellular niche for bacterial growth. To address this issue, we have compared the behaviour of L. monocytogenes in murine bone marrow-derived DC and macrophages (BMM). Similar to BMM, bacteria escaped to the cytosol in DC, replicated, and spread to adjacent cells. However, DC infection was less robust in terms of intracellular doubling time and total increase in bacterial numbers. Immunofluorescence analysis using a strain of L. monocytogenes that expresses green fluorescent protein upon bacterial entry into the cytosol suggested that a subpopulation of DC restricted bacteria to vacuoles, a finding that was confirmed by electron microscopy. In unstimulated DC cultures, L. monocytogenes replicated preferentially in phenotypically immature cells. Furthermore, DC that were induced to mature prior to infection were poor hosts for bacterial growth. We conclude that DC provide a suboptimal niche for L. monocytogenes growth, and this is at least in part a function of the DC maturation state. Therefore, the generation of an effective T cell response may be a net effect of both productive and non-productive infection of DC.

Inhibition of host-directed gene expression by the matrix (M) protein of vesicular stomatitis virus (VSV) effectively blocks host antiviral responses, promotes virus replication, and disables the host cell. However, dendritic cells (DC) have the capacity to resist these effects and remain functional during VSV infection. Here, the mechanisms of DC resistance to M protein and their subsequent maturation were addressed. Flt3L-derived murine bone marrow dendritic cells (FDC), which phenotypically resemble resident splenic DC, continued to synthesize cellular proteins and matured during single-cycle (high-multiplicity) and multicycle (low-multiplicity) infection with VSV. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived myeloid DC (GDC), which are susceptible to M protein effects, were nevertheless capable of maturing, but the response was delayed and occurred only during multicycle infection. FDC resistance was manifested early and was type I interferon (IFN) receptor (IFNAR) and MyD88 independent, but sustained resistance required IFNAR. MyD88-dependent signaling contributed to FDC maturation during single-cycle infection but was dispensable during multicycle infection. Similar to FDC, splenic DC were capable of maturing in vivo during the first 24 h of infection with VSV, and neither Toll-like receptor 7 (TLR7) nor MyD88 was required. We conclude that FDC resistance to M protein is controlled by an intrinsic, MyD88-independent mechanism that operates early in infection and is augmented later in infection by type I IFN. In contrast, while GDC are not intrinsically resistant, they can acquire resistance during multicycle infection. In vivo , splenic DC resist the inhibitory effects of VSV, and as in multicycle FDC infection, MyD88-independent signaling events control their maturation.

Inactivated whole-cell vaccines present a full repertoire of antigens to the immune system. Formalin treatment, a standard method for microbial inactivation, can modify or destroy protein antigenic epitopes. We tested the hypothesis that photochemical inactivation with psoralen and UVA light (PUVA), which targets nucleic acid, would improve the immunogenicity of an Enterotoxigenic E. coli (ETEC) vaccine relative to a formalin-inactivated counterpart. Exposure of ETEC H10407 to PUVA using the psoralen drug 4′-Aminomethyltrioxsalen hydrochloride (AMT) yielded replication-incompetent bacteria that retained their metabolic activity. CFA/I-mediated mannose-resistant hemagglutination (MRHA) was equivalent for PUVA-inactivated and live ETEC, but was severely reduced for formalin–ETEC, indicating that PUVA preserved fimbrial protein functional integrity. The immunogenicity of PUVA–ETEC and formalin–ETEC was compared in mice ± double mutant heat-labile enterotoxin (dmLT) adjuvant. Two weeks after an intramuscular prime/boost, serum anti-ETEC IgG titers were similar for the two vaccines and were increased by dmLT. However, the IgG responses raised against several conserved ETEC proteins were greater after vaccination with PUVA–ETEC. In addition, PUVA–ETEC generated IgG specific for heat-labile toxin (LT) in the absence of dmLT, which was not a property of formalin–ETEC. These data are consistent with PUVA preserving ETEC protein antigens in their native-like form and justify the further testing of PUVA as a vaccine platform for ETEC using murine challenge models.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of lower urinary tract infection (UTI). UTI presents a serious health risk and has considerable secondary implications including economic burden, recurring episodes, and overuse of antibiotics. A safe and effective vaccine would address this widespread health problem and emerging antibiotic resistance. Killed, whole-cell vaccines have shown limited efficacy to prevent recurrent UTI in human trials. We explored photochemical inactivation with psoralen drugs and UVA light (PUVA), which crosslinks nucleic acid, as an alternative to protein-damaging methods of inactivation to improve whole-cell UTI vaccines. Exposure of UPEC to the psoralen drug AMT and UVA light resulted in a killed but metabolically active (KBMA) state, as reported previously for other PUVA-inactivated bacteria. The immunogenicity of PUVA-UPEC as compared to formalin-inactivated UPEC was compared in mice. Both generated high UPEC-specific serum IgG titers after intramuscular delivery. However, using functional adherence as a measure of surface protein integrity, we found differences in the properties of PUVA- and formalin-inactivated UPEC. Adhesion mediated by Type-1 and P-fimbriae was severely compromised by formalin but was unaffected by PUVA, indicating that PUVA preserved the functional conformation of fimbrial proteins, which are targets of protective immune responses. In vitro assays indicated that although they retained metabolic activity, PUVA-UPEC lost virulence properties that could negatively impact vaccine safety. Our results imply the potential for PUVA to improve killed, whole-cell UTI vaccines by generating bacteria that more closely resemble their live, infectious counterparts relative to vaccines generated with protein-damaging methods. IMPORTANCE: Lower urinary tract infection (UTI), caused primarily by uropathogenic Escherichia coli , represents a significant health burden, accounting for 7 million primary care and 1 million emergency room visits annually in the United States. Women and the elderly are especially susceptible and recurrent infection (rUTI) is common in those populations. Lower UTI can lead to life-threatening systemic infection. UTI burden is manifested by healthcare dollars spent (1.5 billion annually), quality of life impact, and resistant strains emerging from antibiotic overuse. A safe and effective vaccine to prevent rUTI would address a substantial healthcare issue. Vaccines comprised of inactivated uropathogenic bacteria have yielded encouraging results in clinical trials but improvements that enhance vaccine performance are needed. To that end, we focused on inactivation methodology and provided data to support photochemical inactivation, which targets nucleic acid, as a promising alternative to conventional protein-damaging inactivation methods to improve whole-cell UTI vaccines.

Recombinant vesicular stomatitis virus (VSV) is a promising platform for vaccine development. M51R VSV, an attenuated, M protein mutant strain, is an effective inducer of Type I interferon and dendritic cell (DC) maturation, which are desirable properties to exploit for vaccine design. We have previously evaluated M51R VSV (M51R) and M51R VSV that produces flagellin (M51R-F) as vaccine vectors using murine models, and found that flagellin enhanced DC activation and VSV-specific antibody production after low-dose vaccination. In this report, the immunogenicity of M51R vectors and the adjuvant effect of virus-produced flagellin were evaluated in nonhuman primates following high-dose (10⁸ pfu) and low-dose (10⁵ pfu) vaccination. A single intramuscular vaccination of African green monkeys with M51R or M51R-F induced VSV-specific, dose-dependent humoral immune responses. Flagellin induced a significant increase in antibody production (IgM, IgG and neutralizing antibody) at the low vaccination dose. A VSV-specific cellular response was detected at 6 weeks post-vaccination, but was neither dose-dependent nor enhanced by flagellin; similar numbers of VSV-specific, IFNγ-producing cells were detected in lymph node and spleen of all animals. These results indicate that virus-directed, intracellular flagellin production may improve VSV-based vaccines encoding heterologous antigens by lowering the dose required to achieve humoral immunity.