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Guinea pig cytomegalovirus (GPCMV) immediate early proteins, IE1 and IE2, demonstrated structural and functional homologies with human cytomegalovirus (HCMV). GPCMV IE1 and IE2 co-localized in the nucleus with each other, the viral polymerase and guinea pig ND10 components (gpPML, gpDaxx, gpSp100, gpATRX). IE1 showed direct interaction with ND10 components by immunoprecipitation unlike IE2. Additionally, IE1 protein disrupted ND10 bodies. IE1 mutagenesis mapped the nuclear localization signal to the C-terminus and identified the core domain for gpPML interaction. Individual knockout of GPCMV GP122 or GP123 (IE2 and IE1 unique exons respectively) was lethal to the virus. However, an IE1 mutant (codons 234–474 deleted), was viable with attenuated viral growth kinetics and increased susceptibility to type I interferon (IFN-I). In HCMV, the IE proteins are important T cell target antigens. Consequently, characterization of the homologs in GPCMV provides a basis for their evaluation in candidate vaccines against congenital infection.

Although a number of antiviral agents are licensed for treatment of some human herpesvirus (HHV) infections, effective antiviral therapy is not available for all HHVs. Additional complications are associated with approved drugs, such as toxicity and side effects, and rise in drug-resistant strains is a driving force for new drug development. Success in HHV vaccine development is limited with only vaccines against varicella-zoster virus currently in use in the clinic. In vitro, in vivo and in silico high-throughput (HTP) approaches and innovative microfluidic systems will provide novel technologies to efficiently identify and evaluate new targets and antiherpetic compounds. Coupled with HTP strategies for manipulation of herpesvirus viral genomes, these strategies will greatly accelerate the development of future antivirals as well as candidate vaccine intervention strategies.The authors provide a brief overview of the herpesvirus family and associated diseases. Further, the authors discuss the approved and investigational antiherpetic drugs in the context of current HTP technologies.HTP technology such as microfluidic systems is crucial for the identification and validation of novel drug targets and next-generation antivirals. Current drug development is limited by the unavailability of HTP preclinical model systems. Specific advancement in the development of HTP animal-specific technology, applied in parallel, allows a more rapid evaluation of drugs at the preclinical stage. The advancement of HTP combinatorial drug therapy, especially 'Organ-on-a-Chip' approaches, will aid in the evaluation of future antiviral compounds and intervention strategies.

Dendritic cells (DCs) are important in linking innate and adaptive immune responses by priming and polarizing naive CD4+ Th cells, but little is known about the effect of different human DC subsets on Th cells, particularly Th17 cells. We have investigated the ability of TLR-stimulated human Langerhans cells (LC), dermal DCs (dDC), and monocyte-derived DCs (moDC) to affect naive and memory Th17 and Th1 responses. MoDCs stimulated greater memory T cell proliferation while LCs and dDCs more potently stimulated naive T cell proliferation, indicating functionally distinct subsets of DCs. TLR stimulation of all three DC types was unable to induce Th17 polarization from naive T cell precursors, despite inducing Th1 polarization. Dectin stimulation of DCs in IMDM was however able to produce Th17 cells. TLR-stimulated DCs were capable of inducing IL-17A and IFN-γ production from memory T cells, although the mechanism used by each DC subset differed. MoDCs partially mediated this effect on memory Th1 and Th17 cells by the production of soluble factors, which correlated with their ability to secrete IL-12p70 and IL-23. In contrast, LCs and dDCs were able to elicit a similar memory response to moDCs, but in a contact dependent manner. Additionally, the influence of microbial stimulation was demonstrated with TLR3 and TLR7/8 agonists inducing a Th1 response, whereas TLR2 or dectin stimulation of moDCs enhanced the IL-17 response. This study emphasizes the differences between human DC subsets and demonstrates that both the DC subset and the microbial stimulus influence the Th cell response.

Dendritic cell (DC) subsets can mediate diverse responses, but little is known about the Toll-like receptor (TLR) signalling pathways in different human DC subsets. Despite expressing many TLRs in common, we found that in vitro-derived Langerhans cells (LCs) and monocyte-derived DCs (moDCs) undergo differential signalling events following TLR stimulation. TLR-stimulated LCs did not secrete interleukin (IL)-12p70 and thus induced a T helper type 2 (Th2)-biased response. moDCs secrete high levels of IL-12p70 and induce a Th1 response. Stimulation of moDCs through TLR2 or TLR7/8 was able to induce phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular-signal-regulated kinase (ERK). However, phosphorylated ERK was not induced in TLR-stimulated LCs, suggesting an ERK-independent method of Th2 cell induction. Inhibition of p38 MAPK suppressed moDC maturation, but was much less effective at inhibiting LC maturation. Phosphatidylinositol-3 kinase (PI3K) was also found to play a greater role in moDC survival compared with the LCs. Polymerase chain reaction (PCR) arrays to compare the expression of signalling molecules in LCs and moDCs identified differences in TLR recognition molecules and cytokine response genes, suggesting that differential functional responses are probably mediated at the post-transcriptional level. Thus we have described differences in LC and moDC responses to TLR stimulation, and have identified key differences in ERK phosphorylation and the involvement of MAPK and PI3K.

In the recently halted HIV type 1 (HIV-1) vaccine STEP trial, individuals that were seropositive for adenovirus serotype 5 (Ad5) showed increased rates of HIV-1 infection on vaccination with an Ad5 vaccine. We propose that this was due to activation and expansion of Ad5-specific mucosal-homing memory CD4 T cells. To test this hypothesis, Ad5 and Ad11 antibody titers were measured in 20 healthy volunteers. Dendritic cells (DCs) from these individuals were pulsed with replication defective Ad5 or Ad11 and co-cultured with autologous lymphocytes. Cytokine profiles, proliferative capacity, mucosal migration potential, and susceptibility to HIV infection of the adenovirus-stimulated memory CD4 T cells were measured. Stimulation of T cells from healthy Ad5-seropositive but Ad11-seronegative individuals with Ad5, or serologically distinct Ad11 vectors induced preferential expansion of adenovirus memory CD4 T cells expressing α 4 β 7 integrins and CCR9, indicating a mucosal-homing phenotype. CD4 T-cell proliferation and IFN-γ production in response to Ad stimulation correlated with Ad5 antibody titers. However, Ad5 serostatus did not correlate with total cytokine production upon challenge with Ad5 or Ad11. Expanded Ad5 and Ad11 memory CD4 T cells showed an increase in CCR5 expression and higher susceptibility to infection by R5 tropic HIV-1. This suggests that adenoviral-based vaccination against HIV-1 in individuals with preexisting immunity against Ad5 results in preferential expansion of HIV-susceptible activated CD4 T cells that home to mucosal tissues, increases the number of virus targets, and leads to a higher susceptibility to HIV acquisition.