Prophylactic Herpes Simplex Virus 2 (HSV-2) Vaccines Adjuvanted with Stable Emulsion and Toll-Like Receptor 9 Agonist Induce a Robust HSV-2-Specific Cell-Mediated Immune Response, Protect against Symptomatic Disease, and Reduce the Latent Viral Reservoir.

Several prophylactic vaccines targeting herpes simplex virus 2 (HSV-2) have failed in the clinic to demonstrate sustained depression of viral shedding or protection from recurrences. Although these vaccines have generated high titers of neutralizing antibodies (NAbs), their induction of robust CD8 T cells has largely been unreported, even though evidence for the importance of HSV-2 antigen-specific CD8 T cells is mounting in animal models and in translational studies involving subjects with active HSV-2-specific immune responses. We developed a subunit vaccine composed of the NAb targets gD and gB and the novel T cell antigen and tegument protein UL40, and we compared this vaccine to a whole-inactivated-virus vaccine (formaldehyde-inactivated HSV-2 [FI-HSV-2]). We evaluated different formulations in combination with several Th1-inducing Toll-like receptor (TLR) agonists in vivo In mice, the TLR9 agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide formulated in a squalene-based oil-in-water emulsion promoted most robust, functional HSV-2 antigen-specific CD8 T cell responses and high titers of neutralizing antibodies, demonstrating its superiority to vaccines adjuvanted by monophosphoryl lipid A (MPL)-alum. We further established that FI-HSV-2 alone or in combination with adjuvants as well as adjuvanted subunit vaccines were successful in the induction of NAbs and T cell responses in guinea pigs. These immunological responses were coincident with a suppression of vaginal HSV-2 shedding, low lesion scores, and a reduction in latent HSV-2 DNA in dorsal root ganglia to undetectable levels. These data support the further preclinical and clinical development of prophylactic HSV-2 vaccines that contain appropriate antigen and adjuvant components responsible for programming elevated CD8 T cell responses.IMPORTANCE Millions of people worldwide are infected with herpes simplex virus 2 (HSV-2), and to date, an efficacious prophylactic vaccine has not met the rigors of clinical trials. Attempts to develop a vaccine have focused primarily on glycoproteins necessary for HSV-2 entry as target antigens and to which the dominant neutralizing antibody response is directed during natural infection. Individuals with asymptomatic infection have exhibited T cell responses against specific HSV-2 antigens not observed in symptomatic individuals. We describe for the first time the immunogenicity profile in animal models of UL40, a novel HSV-2 T cell antigen that has been correlated with asymptomatic HSV-2 disease. Additionally, vaccine candidates adjuvanted by a robust formulation of the CpG oligonucleotide delivered in emulsion were superior to unadjuvanted or MPL-alum-adjuvanted formulations at eliciting a robust cell-mediated immune response and blocking the establishment of a latent viral reservoir in the guinea pig challenge model of HSV-2 infection.

A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation.

The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.

Identification of GLA/SE as an effective adjuvant for the induction of robust humoral and cell-mediated immune responses to EBV-gp350 in mice and rabbits.

Childhood infection with Epstein-Barr virus (EBV) is often asymptomatic and may result in mild flu-like symptoms, but exposure during adolescence and young adulthood can lead to acute infectious mononucleosis (AIM) with a pathology characterized by swollen lymph nodes, sore throat, and severe fatigue lasting weeks or months. A vaccine targeting the envelope glycoprotein gp350 adjuvanted with aluminum hydroxide complexed with the TLR4 agonist monophosphoryl lipid A (MPLA) achieved a 78% reduction in AIM incidence in a small phase II trial of college-age individuals, but development of this vaccine was halted by the manufacturer. Here, we report the evaluation in mice and rabbits of an EBV-gp350 vaccine combined with an adjuvant composed of the synthetic TLR4 agonist glucopyranosyl lipid A (GLA) integrated into stable emulsion (SE). In mice, GLA/SE-adjuvanted gp350 generated high IgG titers (both IgG1 and IgG2a/c subtypes), elevated EBV-neutralizing antibody titers, and robust poly-functional anti-gp350 CD4(+) T cell responses. In addition, GLA/SE routinely demonstrated superior performance over aluminum hydroxide in all immunological readouts, including induction of durable neutralizing antibody titers out to at least 1 year post-vaccination. Both components of the GLA/SE adjuvant were found to be required to get optimal responses in both arms of the immune response: specifically, SE for neutralizing antibodies and GLA for induction of T cell responses. Furthermore, this vaccine also elicited high neutralizing antibody titers in a second species, rabbit. These promising results suggest that clinical development of a vaccine comprised of EBV-gp350 plus GLA/SE has the potential to prevent AIM in post-adolescents.

Negative regulation of TLR9-mediated IFN-alpha induction by a small-molecule, synthetic TLR7 ligand.

Toll-like receptors (TLRs) are a family of molecules that function as sensors for the detection of foreign pathogens through the recognition of nonvariable microbial motifs. Although numerous studies have focused on singular TLRs, less attention has been focused on how simultaneous signaling of multiple TLRs may result in counter-regulation of the effects of each. Here, we examine the counter-regulation that occurs during simultaneous stimulation of TLR7 and TLR9 on human plasmacytoid dendritic cells (PDCs) and B cells. Interestingly, we observed that the capacity for potent IFN-alpha-induction by TLR9 ligands like CpG-C and CpG-A is markedly reduced by concurrent small molecule TLR7 stimulation. However, this inhibition is specific to particular CpG motif-containing immunostimulatory sequence (ISS) functions such as IFN-alpha induction and BDCA-2 down-regulation. Other ISS activities such as PDC expression of CD80/CD86, secretion of IL-6, and B cell proliferation are not altered by the presence of TLR7 ligands (TLR7Ls). In concordance with the ability of TLR7Ls to decrease IFN-alpha secretion induced by ISS, we also find that the expression of interferon regulatory factor-7 (IRF-7), a transcriptional factor critical for IFN-alpha expression, is reduced. Furthermore, down-regulation of TLR9 mRNA expression is accelerated after TLR7 stimulation. These data indicate that TLR7 and TLR9 costimulation do not combine synergistically for IFN-alpha induction and demonstrate that, instead, a negative feedback mechanism has evolved, possibly to prevent levels of IFN-alpha secretion potentially detrimental to the host.

Induction of interferon-gamma from natural killer cells by immunostimulatory CpG DNA is mediated through plasmacytoid-dendritic-cell-produced interferon-alpha and tumour necrosis factor-alpha.

Immunostimulatory sequences (ISS) that contain CpG motifs have been demonstrated to exert antipathogen and antitumour immunity in animal models through several mechanisms, including the activation of natural killer (NK) cells to secrete interferon-gamma (IFN-gamma) and to exert lytic activity. Since NK cells lack the ISS receptor TLR9, the exact pathway by which NK cells are activated by ISS is unclear. We determined that ISS-induced IFN-gamma from NK cells is primarily dependent upon IFN-alpha release from plasmacytoid dendritic cells (PDCs), which directly activates the NK cell. However, further analysis indicated that other PDC-released soluble factor(s) may contribute to IFN-gamma induction. Indeed, tumour necrosis factor-alpha (TNF-alpha) was identified as a significant contributor to ISS-mediated activation of NK cells and was observed to act in an additive fashion with IFN-alpha in the induction of IFN-gamma from NK cells and to up-regulate CD69 expression on NK cells. This activity of TNF-alpha, however, was dependent upon the presence of PDC-derived factors such as type I interferon. These results illustrate an important function for type I interferon in innate immunity, which is not only to activate effectors like NK cells directly, but also to prime them for enhanced activation by other factors such as TNF-alpha.