Multiple tandem copies of conserved gp41 epitopes incorporated in gag virus-like particles elicit systemic and mucosal antibodies in an optimized heterologous vector delivery regimen.

Induction of neutralizing antibodies to prevent HIV infection, especially at the mucosa, is a critical goal of future vaccines. In this study, we have designed chimeric HIV-gag virus-like particles (VLPs) that contain multiple copies of the two highly conserved gp41 membrane-proximal external region (MPER) epitopes, ELDKWA and NWFDIT, with the objective of generating high titers of MPER-specific antibodies. We have shown that the implementation of optimized vector design, delivery regimens and appropriate delivery methods is critical to significantly increase epitope-specific antibody titers. One goal of the methods that were tested and employed was to generate high levels of mucosal MPER-specific antibodies, as mucosal immune induction could play a key role in preventing HIV infection. We also tested a design strategy that incorporated multiple repeats of the MPER epitopes within gag, which significantly increased specific antibody titers, systemically and mucosally. This alternative design strategy and the implementation of optimized heterologous immunization regimens can serve to 'immuno-focus' and significantly increase epitope-specific titers.

Treatment of intravaginal HSV-2 infection in mice: a comparison of CpG oligodeoxynucleotides and resiquimod (R-848).

The mammalian innate immune system recognizes pathogens via a series of pattern-recognition receptors such as the toll-like receptors (TLR) that interact with pathogen-associated molecular patterns (PAMPs) and lead to the rapid activation of innate immune cells. In this study, we compared the efficacy of CpG ODN (a TLR9 agonist) and resiquimod (R-848; a TLR7/8 agonist) for topical immunoprophylaxis or immunotherapy of vaginal herpes simplex virus type 2 (HSV-2) infection in mice. Efficacy against HSV infection was observed with CpG ODN but less so with R-848, even after repeated administrations. Intravaginal (IVAG) administration of CpG ODN resulted in strong local but relatively weak systemic immune activation, as determined by levels of the chemokines IP-10, MIG and I-TAC in vaginal tissue and plasma, respectively. In contrast, IVAG administration of R-848 resulted in high levels of plasma IP-10, similar to those seen after parenteral administration, but overall, weaker or shorter-lived local immune responses than obtained with CpG ODN. These findings suggest that differences in biodistribution and sites of immune activation between CpG ODN and R-848 after IVAG delivery account for differences in efficacy, and demonstrate the need for local mucosal innate activation for protection against HSV-2.

Mucosal delivery of CpG oligodeoxynucleotides expands functional dendritic cells and macrophages in the vagina.

Antigen-presenting cells (APC) are specialized sentinel cells that sense pathogens within tissues and then activate appropriate immune effector cells in lymphoid organs. Recent evidence, however, suggests that APC can also induce effector cells in non-lymphoid organs. The purpose of this study was to determine the effect of intravaginal (IVAG) delivery of CpG-oligodeoxynucleotide (ODN) on expansion of resident genital APC. Our results show that delivery of CpG-ODN to the murine genital tract induced a rapid and significant, but transient expansion of genital APC in situ. As early as 12 hr post CpG-ODN delivery, we observed an enhanced level of F4/80+ major histocompatibility complex (MHC) class II-negative macrophages in the genital tissue. This was followed by increased levels of F4/80/MHC class II double-positive cells, as well as MHC class II, CD11c and CD86 triple-positive dendritic cells (DC) at 48 hr. Expanded APC levels at 48 hr post CpG-ODN resulted in increased ability of genital cells to induce an allogenic mixed leucocyte reaction. By 72 hr after CpG-ODN treatment, APC levels were not distinguishable from naive levels. Therefore, these results clearly show that administration of CpG-ODN to the genital tract induced a marked but transient enhancement of APC within the genital tissue, and that these APC appear to possess functional capacity. Furthermore, these results indicate that IVAG-CpG-ODN may be an important factor for the enhancement of local antigen presentation in the genital tract through increased DC numbers.

Toll-like receptor (TLR)-3, but not TLR4, agonist protects against genital herpes infection in the absence of inflammation seen with CpG DNA.

We previously demonstrated that delivery of CpG oligodeoxynucleotide (ODN) to vaginal mucosa induced an innate mucosal antiviral state that protected against intravaginal challenge with herpes simplex virus (HSV)-2. We report that mucosal, but not systemic, delivery of ligands for Toll-like receptor (TLR)-3, but not TLR4, induced protection against genital HSV-2 challenge that was not accompanied by the local inflammation and splenomegaly seen after treatment with CpG ODN. Surprisingly, TLR4 messenger (m) RNA expression was shown to be higher than that of TLR3 or TLR9 in murine genital mucosa. Similarly, murine RAW264.7 cells were shown to express more mRNA for TLR4 than TLR3 or TLR9, yet treatment of these cells with double-stranded RNA provided greater protection than lipopolysaccharide or CpG ODN. These results indicate that TLR3 ligand induces a more potent antiviral response than TLR4 and TLR9 ligands and may be a safer means of protecting against sexually transmitted viral infections.

Parameters of CpG oligodeoxynucleotide-induced protection against intravaginal HSV-2 challenge.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides within the context of certain flanking bases (CpG motifs) have been shown to induce potent innate and adaptive immune responses. Vaginal delivery of CpG ODN alone protects mice from vaginal herpes simplex virus type-2 (HSV-2) challenge. Here, we investigated the importance of timing of delivery, formulation, route and dose of vaginally administered CpG ODN in the prevention or treatment of intravaginal (IVAG) HSV-2 infection. Mice treated intravaginally with CpG ODN containing a phosphorothioate backbone 24 hours prior to IVAG HSV-2 challenge survived infection, showed minimal vaginal pathology, and had virtually no detectable virus in vaginal washes, when compared to mice treated with non-CpG ODN. Genital treatment of HSV-2 infected mice with CpG ODN 4 hours after infection resulted in increased survival and decreased pathology and vaginal virus titers, whereas treatment of infected mice with CpG ODN 24 and 72 hours after IVAG HSV-2 infection had no effect on disease progression. Both liquid and solid (delivered on a bio-erodible muco-adhesive film) formulations of CpG ODN were effective in protection against genital HSV-2 following vaginal delivery. Lastly, IVAG delivery of 10 micro g of CpG ODN protected as well as a 100 micro g dose.