The role of Type I interferon (IFN) during pathogenic HIV and SIV infections remains unclear, with conflicting observations suggesting protective versus immunopathological effects. We therefore examined the effect of IFNα/β on T cell death and viremia in HIV infection. Ex vivo analysis of eight pro- and anti-apoptotic molecules in chronic HIV-1 infection revealed that pro-apoptotic Bak was increased in CD4+ T cells and correlated directly with sensitivity to CD95/Fas-mediated apoptosis and inversely with CD4+ T cell counts. Apoptosis sensitivity and Bak expression were primarily increased in effector memory T cells. Knockdown of Bak by RNA interference inhibited CD95/Fas-induced death of T cells from HIV-1-infected individuals. In HIV-1-infected patients, IFNα-stimulated gene expression correlated positively with ex vivo T cell Bak levels, CD95/Fas-mediated apoptosis and viremia and negatively with CD4+ T cell counts. In vitro IFNα/β stimulation enhanced Bak expression, CD95/Fas expression and CD95/Fas-mediated apoptosis in healthy donor T cells and induced death of HIV-specific CD8+ T cells from HIV-1-infected patients. HIV-1 in vitro sensitized T cells to CD95/Fas-induced apoptosis and this was Toll-like receptor (TLR)7/9- and Type I IFN-dependent. This sensitization by HIV-1 was due to an indirect effect on T cells, as it occurred in peripheral blood mononuclear cell cultures but not purified CD4+ T cells. Finally, peak IFNα levels and viral loads correlated negatively during acute SIV infection suggesting a potential antiviral effect, but positively during chronic SIV infection indicating that either the virus drives IFNα production or IFNα may facilitate loss of viral control. The above findings indicate stage-specific opposing effects of Type I IFNs during HIV-1 infection and suggest a novel mechanism by which these cytokines contribute to T cell depletion, dysregulation of cellular immunity and disease progression.

A significant hurdle in vaccine development for many infectious pathogens is the ability to generate appropriate immune responses at the portal of entry, namely mucosal sites. The development of vaccine approaches resulting in secretory IgA and mucosal cellular immune responses against target pathogens is of great interest and in general, requires live viral infection at mucosal sites. Using HIV-1 and influenza A antigens as models, we report here that a novel systemically administered DNA vaccination strategy utilizing co-delivery of the specific chemokine molecular adjuvant CCL25 (TECK) can produce antigen-specific immune responses at distal sites including the lung and mesenteric lymph nodes in mice. The targeted vaccines induced infiltration of cognate chemokine receptor, CCR9+/CD11c+ immune cells to the site of immunization. Furthermore, data shows enhanced IFN-λ secretion by antigen-specific CD3+/CD8+ and CD3+/CD4+ T cells, as well as elevated HIV-1-specific IgG and IgA responses in secondary lymphoid organs, peripheral blood, and importantly, at mucosal sites. These studies have significance for the development of vaccines and therapeutic strategies requiring mucosal immune responses and represent the first report of the use of plasmid co-delivery of CCL25 as part of the DNA vaccine strategy to boost systemic and mucosal immune responses following intramuscular injection.

There have been encouraging results for the development of an effective HIV vaccine. However, many questions remain regarding the quality of immune responses and the role of mucosal antibodies. We addressed some of these issues by using a simian immunodeficiency virus (SIV) DNA vaccine adjuvanted with plasmid-expressed mucosal chemokines combined with an intravaginal SIV challenge in rhesus macaque (RhM) model. We previously reported on the ability of CCR9 and CCR10 ligand (L) adjuvants to enhance mucosal and systemic IgA and IgG in small animals. In this study, RhMs were intramuscularly immunized five times with either DNA or DNA plus chemokine adjuvant delivered by electroporation followed by challenge with SIVsmE660. Sixty-eight percent of all vaccinated animals (P=0.0016) remained either uninfected or had aborted infection compared to only 14% in the vaccine naïve group. The highest protection was observed in the CCR10L chemokines group, where 6 of 9 animals had aborted infection and two remained uninfected, leading to 89% protection (P=0.0003). The induction of mucosal SIV-specific antibodies and neutralization titers correlated with trends in protection. These results indicate the need to further investigate the contribution of chemokine adjuvants to modulate immune responses and the role of mucosal antibodies in SIV/HIV protection.

No abstract

Development of a vaccine that drives anti-viral mucosal B cell responses is critical for protection against HIV-1 infection. It is currently unknown whether triggering CCR10/CCL28 pathways in a DNA-based vaccine results in induction of HIV-1env specific B cell immunity at mucosal sites of infection. We hypothesized that co-immunization with HIV-1env/CCL28 molecular adjuvant would augment B cell responses at gastrointestinal and vaginal sites and require CCR10. CCL28 co-immunized WT mice displayed a significant enhancement of HIV-1env specific antibody titers in serum, feces and vaginal washes, and enhanced HIV-1 specific IgA responses were abrogated in CCR10KO mice. CCL28 co-immunization did not increase the breadth of linear B cell epitopes in WT mice, but augmented the dominant epitopes elicited by antigen immunization alone. The frequency of splenic and intestinal IgA+CD19+B220+CD138+CCR10+ plasmablasts was augmented in the CCL28 co-immunized WT mice over antigen-only immunized WT controls. The physiological relevance of these findings was confirmed in a NHP model of intravaginal SIVsmE660 challenge in which CCL28 co-immunization resulted in significant increases in serum/vaginal IgG/IgA, decrease in peak viral loads, significant suppression of viral titers over 120 days, and recovery of CD4 T cells. These data support a role for CCL28 in targeting protective anti-viral B cells to mucosal sites when delivered as molecular adjuvants for HIV-1env DNA-based vaccines.

We have shown previously that CCR2 is expressed on highly differentiated human Th memory cells as well as mucosal associated invariant T (MAIT) cells. CCR2+ T cells typically co-express a combination of other inflammation-associated chemokine receptors, including CCR6, which is found on all type 17 T cells. We found that the CCR6+ CCR2+ human Th cell population is enriched in cells with a pathogenic cytokine and cytotoxic profile. Single-cell analysis revealed two subtypes of such CCR6+ CCR2+ cells, either biased to produce GM-CSF or IFNg. Pathogenic activity requires migration into inflamed tissue, and to understand the roles for CCR2, CCR6 and other chemokine receptors expressed on these cells in extravasation we used flow chambers containing HUVEC activated with TNFa and/or IFNg. We found that although CCR6, CCR5 and CXCR3 (but not CCR2) were capable of mediating T cell arrest, only CCR2 was active in transendothelial migration. CCR2’s activity was a function of the localization of its principal ligand, CCL2, in endothelial cells. Native CCL2 was found intracellularly but not on the cell surface. Immediate pre-treatment of cells with CCL2 or using HUVEC transduced to express a CCL2-CXCL9 chimeric chemokine that bound to surface glycosaminoglycans enabled CCR2 to mediate T cell arrest. These studies reveal the integration of transendothelial trafficking and a pathogenic effector profile in type 17 human Th cells and demonstrate redundant and non-redundant roles for the multiple chemokine receptors on these cells due to chemokine positioning. Our findings could inform more effective combinatorial inhibition or expression of chemokine receptors/ligands to regulate the migration of highly inflammatory T cells. Supported by NIH

Mucosal antibody plays a vital role in protection against HIV transmission. Mucosa-associated epithelial chemokine (CCL28) is secreted by mucosal epithelial cells following commensal colonization, and binds CC-chemokine receptor 10 (CCR10), expressed on IgA+ plasma cells. Deletion of CCR10 impairs mucosal IgA production and memory responses. Thus, the CCL28/CCR10 axis is critical for regulating mucosal IgA production and memory immunity. Mice were vaccinated twice with consensus HIV-1 gp160 DNA alone (antigen-only), or antigen and plasmid-encoded CCL28 (pCCL28) via intramuscular injection and in vivo electroporation (EP). pCCL28 increased antigen-specific IgA in fecal extracts (average 36.7ng/ml IgA in the antigen only group compared with 90.9ng/ml in the pCCL28 group). The intestinal compartment from animals co-immunized with pCCL28 harbored a higher frequency of HIV-specific, IgA+ B cells compared to the antigen-alone group (13.1% compared to 5.7%, respectively), and these cells expressed significantly-higher levels of CCR10 than those from mice in the non-adjuvanted group (MFI 413 compared to 181, respectively). Colonization with commensals induces CCL28 secretion by intestinal epithelial cells. Indeed, In vitro experiments with human colonic epithelial cell lines indicate that these organisms increased expression of CCL28 at the transcript and protein level. These studies suggest that electroporated delivery of the mucosal chemokine CCL28, can enhance antigen-specific anti-HIV immunity in the mucosa. Ongoing studies will determine if supplementation of commensal flora during vaccination can enhance CCL28 secretion in vivo and promote retention of vaccine-induced IgA+ B cells at mucosal surfaces.

Leukocyte extravasation is a multistep process characterized by rolling and arrest on the endothelium followed by transendothelial migration (TEM). Arrest and TEM by T cells typically requires the activities of chemokines and their receptors. Previously, we showed that CCR2 identifies highly differentiated, readily activated CD4+ memory T cells in human blood with diverse effector capabilities, and we showed that CCR6 is the signature chemokine receptor for Th17 cells. Here we describe studies of the migratory capabilities and effector profiles of CD4+ CCR6+ CCR2+ as compared with CD4+ CCR6+ CCR2− and CD4+ CCR6− CCR2− T cells from human blood. Using flow chamber assays with TNFa-activated human umbilical vein endothelial cells, we found that among the CCR6+ cells, only the CCR2+ subset was able to cross the endothelial monolayer efficiently. Using antibodies against the CCR6 ligand, CCL20, and a specific inhibitor of CCR2, we found that CCR6 was important for arrest and that CCR2 was critical for subsequent TEM. Using intracellular staining of cells activated ex vivo, we found that although IL-17-expressing cells were found within all CCR6+ subsets, the CCR6+CCR2+ cells were distinguished by their production of IFNg, GM-CSF, and TNFa, thereby demonstrating the cytokine profile of pathogenic Th17 cells as identified in mouse models of autoimmune disease. Taken together, our studies suggest that chemokine receptors on memory Th cells play non-redundant roles in the egress from blood, that providing T cells with CCR6 and CCR2 could enhance their trafficking into tissue, and that, on the other hand, blocking CCR2 alone could prevent the extravasation of pathogenic Th17 cells and ameliorate tissue damage in Th17-cell driven disease.