It is believed that an effective HCV vaccine must induce strong HCV-specific cytotoxic IFNγ+ CD8+ T cells able to migrate into and become fully activated within the liver, an organ known to suppress T-cell responses and induce tolerance. Given the importance of intrahepatic HCV-specific T cells in the clearance of acute infection, the goal of this present study was to determine if peripheral immunization was able to induce functional intrahepatic HCV-specific T cell-based immunity both in the presence and absence of HCV antigen expression within the liver. Using a novel HCV NS3/NS4A DNA vaccine, we show that peripheral immunization of C57BL/6 mice results in the formation of a large pool of fully functional HCV-specific cytotoxic IFNγ+ CD8+ T cells within the liver and that these cells were highly enriched within the liver as compared with the spleen. Following hepatic expression of cognate HCV antigen using a previously described liver transfection method, we show that this pool of vaccine-induced HCV-specific CD8+ T cells retained its ability to become highly activated as shown by the upregulation of IFNγ and CCR5 expression, as well as by the clearance of HCV NS3 expressing hepatocytes. Taken together, these findings suggest that T-cell effector function is preserved within the liver and that selective recruitment of antigen-specific T cells to the liver may play a previously unappreciated role in the process of immune surveillance, which may be exploited for future T cell-based HCV vaccines.
HCV DNA vaccine; NS3; NS4A; liver transfection; consensus
Protection against infection is the hallmark of immunity and the basis of effective vaccination. For a variety of reasons there is a great demand to develop new, safer and more effective vaccine platforms. In this regard, while ‘first-generation’ DNA vaccines were poorly immunogenic, new genetic ‘optimization’ strategies and the application of in vivo electroporation (EP) have dramatically boosted their potency. We developed a highly optimized plasmid DNA vaccine that expresses the lymphocytic choriomeningitis virus (LCMV) nucleocapsid protein (NP) and evaluated it using the LCMV challenge model, a gold standard for studying infection and immunity. When administered intramuscularly with EP, robust NP-specific cellular and humoral immune responses were elicited, the magnitudes of which approached those following acute LCMV infection. Furthermore, these responses were capable of providing 100% protection against a high-dose, normally lethal virus challenge. This is the first non-infectious vaccine conferring complete protective immunity up to eight weeks after vaccination and demonstrates the potential utility of ‘next-generation’ DNA vaccines.
DNA vaccine; electroporation; protection; LCMV; T cell; antibody
Early DNA vaccines were very exciting in small animals, but poorly immunogenic in large animals and humans. Recently, much progress has been made regarding increasing their immune potency. This review summarizes many of these technological advancements and discusses the current status and prospects of DNA vaccines in the clinic targeting specific pathogens.
It was discovered almost 20 years ago that plasmid DNA, when injected into the skin or muscle of mice, could induce immune responses to encoded antigens. Since that time, there has since been much progress in understanding the basic biology behind this deceptively simple vaccine platform and much technological advancement to enhance immune potency. Among these advancements are improved formulations and improved physical methods of delivery, which increase the uptake of vaccine plasmids by cells; optimization of vaccine vectors and encoded antigens; and the development of novel formulations and adjuvants to augment and direct the host immune response. The ability of the current, or second-generation, DNA vaccines to induce more-potent cellular and humoral responses opens up this platform to be examined in both preventative and therapeutic arenas. This review focuses on these advances and discusses both preventive and immunotherapeutic clinical applications.
Costimulatory molecules play a central role in the development of cellular immunity. Understanding how costimulatory pathways can be directed to positively influence the immune response may be critical for the generation of an effective HIV vaccine. Here, we evaluated the ability of intravenous administration of a blocking monoclonal antibody (mAb) directed against the negative costimulatory molecule CTLA-4, and an agonist mAb directed against the positive costimulatory molecule 4-1BB, either alone or in combination, to augment intramuscular SIV DNA immunizations. We then tested the ability these of these responses to impact a high-dose SIVmac251 challenge. Following immunization, the groups infused with the anti-4-1BB mAb exhibited enhanced IFN-γ responses compared to the DNA vaccine only group. Interestingly, although CTLA-4 blockade alone did not enhance IFN-γ responses it did increase the proliferative capacity of the CD4+ and CD8+ T cells. The combination of both mAbs enhanced the magnitude of the polyfunctional CD8+ T cell response. Following challenge, the group that received both mAbs exhibited a significant, ∼2.0 log, decrease in plasma viral load compared to the naïve group the included complete suppression of viral load in some animals. Furthermore, the use of the CTLA-4 blocking antibody resulted in significantly higher viral loads during chronic infection compared to animals that received the 4-1BB mAb, likely due to the higher CD4+ T cell proliferative responses which were driven by this adjuvant following immunization. These novel studies show that these adjuvants induce differential modulation of immune responses, which have dramatically different consequences for control of SIV replication, suggesting important implications for HIV vaccine development.
HIV infection and subsequent antiretroviral therapy have been associated with an increased incidence of dyslipidemia and cardiovascular disease and has been shown to suppress cholesterol efflux from virus-infected macrophages by inducing Nef-dependent downregulation of ABCA1. The SIV/macaque model was used to examine consequences and mechanisms involved. SIV infection drove a significant remodeling of high-density lipoprotein profiles suggesting systemic inhibition of the ABCA1-dependent reverse cholesterol transport pathway. The ABCA1 cholesterol transporter was significantly down regulated in the livers of the SIV-infected macaques and the viral protein Nef could be detected in the liver as well as in plasma of infected animals. Extracellular myristoylated HIV Nef inhibited cholesterol efflux from macrophages and hepatocytes. Moreover, sera from SIV-infected macaques also suppressed cholesterol efflux in a Nef-dependent fashion. These results indicate that SIV infection is a significant contributor to primary dyslipidemia, likely through the ability of Nef to suppress ABCA1-dependent reverse cholesterol transport.
HIV; SIV; ABCA1; Nef; atherosclerosis
Adjuvant compounds are usually included in vaccinations in order to bolster total vaccine-specific responses or to tailor an immune response toward a desired endpoint, such as the production of gamma interferon or an increase in antibody titers. While most adjuvants are studied in regard to their impact on vaccine-specific responses during and just after the vaccination period, a detailed analysis of how adjuvants skew the Th1/Th2 axis at more distant time points is not often undertaken. In the current study, we present data that suggests that adjuvants differ in their relative abilities to bolster and skew immune responses in the short term compared with more distant time points. To that end, we have employed interleukin-12 (IL-12) and IL-28B as adjuvants for DNA vaccination of rhesus macaques. While both adjuvants were able to bolster Th1-biased responses, our analysis shows that this skewing was achieved through different mechanisms. Moreover, analysis 3 months after the final immunization revealed the activity of the IL-12 adjuvant to be short lived, while the IL-28B adjuvant continued to exert its influence on the immune system. Taken together, these data suggest that the scientific and medical communities would benefit from a more detailed analysis of adjuvant function, including the determination of long-term influences of administered adjuvants.
The capacity for robust proliferation upon re-infection is a hallmark of adaptive immunity and the basis of vaccination. A widely used animal model for the study of human disease is the rhesus macaque (RM), where capacity for proliferation can be assessed ex vivo using carboxyfluorescein succinimidyl ester (CFSE)-based dilution assays. However, we show over the course of the standard ex vivo proliferation assay that CFSE-labeling at commonly-used dye concentrations induces significant cell death, but that this phenomenon is dose-dependent. Here we describe an alternative, semi-quantitative method for estimating T cell proliferative responses that avoids the putative biases associated with chemical modification. RM peripheral blood mononuclear cells were stimulated ex vivo with cognate peptides for five days, immunostained for intracellular Ki-67, and then analyzed by flow cytometry. We describe a gating strategy using Ki-67 and side light scatter, also a marker of blastogenesis, which correlates strongly with data from CFSE dilution. We show that this method is a valid tool for measuring RM antigen-specific cellular proliferation ex vivo and can be used as an alternative to CFSE dilution assays.
T cell; Ki-67; CFSE; CFDA-SE; rhesus macaque; proliferation assay
We have identified a subset of HIV-susceptible CD4+CCR5+ cells in human PBMCs that can efficiently exclude protease inhibitors (PI) due to high P-glycoprotein (P-gp) efflux activity. Phenotypically these cells are heterogeneous, include both T and non-T cells, and some display markers of memory cells. Cells with high P-gp represent 16–56% (median = 37.3) of all CD4+CCR5+ cells in healthy donors, and are selectively depleted in HIV-1-infected individuals (4.1–33%, median = 10.1). A fraction of primary cells productively infected by HIV-1, in vitro, have high P-gp pump activity, demonstrating that infection does not inhibit P-gp function. In agreement with these data, HIV-susceptible cells expressing high levels of P-gp require higher levels of PI for complete inhibition of virus spread. We conclude that the PI concentrations achieved in plasma could be suboptimal for full inhibition of virus spread in high P-gp cells, indicating that they may represent a pharmacological sanctuary for HIV-1.
Plasmid-encoded DNA vaccines appear to be a safe and effective method for delivering antigen; however, the immunogenicity of such vaccines is often suboptimal. Cytokine adjuvants including interleukin (IL)-12, RANTES, granulocyte-macrophage colony-stimulating factor, IL-15, and others have been used to augment the immune response against DNA vaccines. In particular, IL-15 binds to a unique high-affinity receptor, IL-15Rα; is trans-presented to CD8+ T cells expressing the common βγ chain; and has been shown to play a role in the generation, maintenance, and proliferation of antigen-specific CD8+ T cells. In this study, we took the unique approach of using both a cytokine and its receptor as an adjuvant in an HIV-1 vaccine strategy. To study IL-15Rα expression, a unique monoclonal antibody (KK1.23) was generated to confirm receptor expression in vitro. Coimmunization of IL-15 and IL-15Rα plasmids with HIV-1 antigenic plasmids in mice enhanced the antigen-specific immune response 2-fold over IL-15 immunoadjuvant alone. Furthermore, plasmid-encoded IL-15Rα augments immune responses in the absence of IL-15, suggesting its role as a novel adjuvant. Moreover, pIL-15Rα enhanced the cellular, but not the humoral, immune response as measured by antigen-specific IgG antibody. This is the first report describing that IL-15Rα itself can act as an adjuvant by enhancing an antigen-specific T cell response. Uniquely, pIL-15 and pIL-15Rα adjuvants combined, but not the receptor α chain alone, may be useful as a strategy for generating and maintaining memory CD8+ T cells in a DNA vaccine.
Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging.
Peripheral blood CD27+ B cells are reduced in HIV-1-infected individuals. In healthy individuals, the human peripheral blood CD27+ B cell pool consists of two subsets defined by the expression, or lack thereof, of the CD45 isoform B220. We investigated the presence of circulating B220+ and B220− memory B cells in HIV+ individuals and found that the reduction in CD27+ memory B cells occurs primarily among CD27+B220− B cells. Studies conducted using healthy controls indicate that CD27+B220− B cells have a splenic marginal zone like the immunophenotype IgMhiIgDloCD21+CD23−, express TLR9, and proliferate and secrete IgG and IgM in response to B cell-specific ODN. CD27+B220+ B cells have the immunophenotype IgMloIgDhiCD21+CD23+, express activation-induced cytidine deaminase, and proliferate in response to SAC but do not secrete immunoglobulin. The AICD expression, along with CD86 expression, by CD27+B220+ suggests these cells are of germinal center origin. The preferential depletion of CD27+B220− B cells mirrors alterations in spleen morphology and resident B cell populations due to HIV infection reported by other investigators and may play an important role in the defective B cell immunity against T-independent pathogens such as pneumococcus observed in HIV-1-infected individuals.
Several steps of HIV-1 replication critically depend on cholesterol. HIV infection is associated with profound changes in lipid and lipoprotein metabolism and an increased risk of coronary artery disease. Whereas numerous studies have investigated the role of anti-HIV drugs in lipodystrophy and dyslipidemia, the effects of HIV infection on cellular cholesterol metabolism remain uncharacterized. Here, we demonstrate that HIV-1 impairs ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux from human macrophages, a condition previously shown to be highly atherogenic. In HIV-1–infected cells, this effect was mediated by Nef. Transfection of murine macrophages with Nef impaired cholesterol efflux from these cells. At least two mechanisms were found to be responsible for this phenomenon: first, HIV infection and transfection with Nef induced post-transcriptional down-regulation of ABCA1; and second, Nef caused redistribution of ABCA1 to the plasma membrane and inhibited internalization of apolipoprotein A-I. Binding of Nef to ABCA1 was required for down-regulation and redistribution of ABCA1. HIV-infected and Nef-transfected macrophages accumulated substantial amounts of lipids, thus resembling foam cells. The contribution of HIV-infected macrophages to the pathogenesis of atherosclerosis was supported by the presence of HIV-positive foam cells in atherosclerotic plaques of HIV-infected patients. Stimulation of cholesterol efflux from macrophages significantly reduced infectivity of the virions produced by these cells, and this effect correlated with a decreased amount of virion-associated cholesterol, suggesting that impairment of cholesterol efflux is essential to ensure proper cholesterol content in nascent HIV particles. These results reveal a previously unrecognized dysregulation of intracellular lipid metabolism in HIV-infected macrophages and identify Nef and ABCA1 as the key players responsible for this effect. Our findings have implications for pathogenesis of both HIV disease and atherosclerosis, because they reveal the role of cholesterol efflux impairment in HIV infectivity and suggest a possible mechanism by which HIV infection of macrophages may contribute to increased risk of atherosclerosis in HIV-infected patients.
HIV1-Nef impairs ABCA1-dependent cholesterol efflux from infected macrophages, promoting the transformation of virally infected macrophages into foam cells (a condition that may put HIV patients at risk for atherosclerosis).