Article

Modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with DNA immunogens

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Abstract

Immunization with nucleic acids has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. We hypothesize that immunization with DNA could be enhanced by directing specific immune responses induced by the vaccine based on the differential correlates of protection known for a particular pathogen. Recently we and others reported that specific immune responses generated by DNA vaccine could be modulated by co-delivery of gene expression cassettes encoding for IL-12, granulocyte-macrophage colony-stimulating factor and the co-stimulatory molecule CD86. To further engineer the immune response in vivo, we investigated the induction and regulation of immune responses following the co-delivery of pro-inflammatory cytokine (IL-1α, TNF-α, and TNF-β), Th1 cytokine (IL-2, IL-12, IL-15, and IL-18), and Th2 cytokine (IL-4, IL-5 and IL-10) genes. We observed enhancement of antigen-specific humoral response with the co-delivery of Th2 cytokine genes IL-4, IL-5, and IL-10 as well as those of IL-2 and IL-18. A dramatic increase in antigen-specific T helper cell proliferation was seen with IL-2 and TNF-α gene co-injections. In addition, we observed a significant enhancement of the cytotoxic response with the co-administration of TNF-α and IL-15 genes with HIV-1 DNA immunogens. These increases in CTL response were both MHC class I restricted and CD8+ T cell dependent. Together with earlier reports on the utility of co-immunizing using immunologically important molecules together with DNA immunogens, we demonstrate the potential of this strategy as an important tool for the development of more rationally designed vaccines.

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... One of the advantages of genetic vaccines is they can induce humoral and cell enhanced Th1 responses (29,94,96). To enhance Th2 responses, the co-administration of IL-4, IL-5 and IL-10 were effective (29,96). ...
... One of the advantages of genetic vaccines is they can induce humoral and cell enhanced Th1 responses (29,94,96). To enhance Th2 responses, the co-administration of IL-4, IL-5 and IL-10 were effective (29,96). Co-injection of plasmids encoding CD80 and CD86, two co-stimulatory molecules important for T cell activation, also modulates the immune response to the target antigen. ...
... 2,5,[7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] However, delivery of DNA with either of these techniques is not ideal for large scale vaccination programs, particularly in underdeveloped countries. DNA expressed adjuvants, such as cytokines [22][23][24][25][26] or chemokines, [27][28][29] have also been used to boost immunogenicity. Unfortunately, like DNA vaccination itself, many of these adjuvants did not translate well from mice to primates. ...
... Most DNA vaccine adjuvants tested to date have been cytokines or chemokines. [22][23][24][25][26] Unfortunately, most of these adjuvants have not translated well from mice to primates. [30][31][32]91 We and others have exploited the inherent adjuvant activities of CT and LT for use as plasmid DNA expressed adjuvants. ...
Article
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DNA encoded adjuvants are well known for increasing the magnitude of cellular and/or humoral immune responses directed against vaccine antigens. DNA adjuvants can also tune immune responses directed against vaccine antigens to better protect against infection of the target organism. Two potent DNA adjuvants that have unique abilities to tune immune responses are the catalytic A1 domains of Cholera Toxin (CTA1) and Heat-Labile Enterotoxin (LTA1). Here, we have characterized the adjuvant activities of CTA1 and LTA1 using HIV and SIV genes as model antigens. Both of these adjuvants enhanced the magnitude of antigen-specific cellular immune responses on par with those induced by the well-characterized cytokine adjuvants IL-12 and GM-CSF. CTA1 and LTA1 preferentially enhanced cellular responses to the intracellular antigen SIVmac239-gag over those for the secreted HIVBaL-gp120 antigen. IL-12, GM-CSF and electroporation did the opposite suggesting differences in the mechanisms of actions of these diverse adjuvants. Combinations of CTA1 or LTA1 with IL-12 or GM-CSF generated additive and better balanced cellular responses to both of these antigens. Consistent with observations made with the holotoxin and the CTA1-DD adjuvant, CTA1 and LTA1 evoked mixed Th1/Th17 cellular immune responses. Together, these results show that CTA1 and LTA1 are potent DNA vaccine adjuvants that favor the intracellular antigen gag over the secreted antigen gp120 and evoke mixed Th1/Th17 responses against both of these antigens. The results also indicate that achieving a balanced immune response to multiple intracellular and extracellular antigens delivered via DNA vaccination may require combining adjuvants that have different and complementary mechanisms of action.
... Many molecular adjuvants have been used in combination with DNA vaccines. To mention few most prominent ones which increased vaccine immunogenicity and induced sustained memory response: IL-2 (Kim et al. 1998(Kim et al. , 2000, IL-12 (Chattergoon et al. 2004;Halwani et al. 2008;Naderi et al. 2013), and GM-CSF (Weiss et al. 1998;Ahlers et al. 2002;Yoon et al. 2006). For a more comprehensive review on molecular adjuvants, see Laddy and Weiner (2006), Abdulhaqq and Weiner (2008), and Suschak et al. (2017). ...
Article
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Inactivated and live attenuated vaccines have improved human life and significantly reduced morbidity and mortality of several human infectious diseases. However, these vaccines have faults, such as reactivity or suboptimal efficacy and expensive and time-consuming development and production. Additionally, despite the enormous efforts to develop vaccines against some infectious diseases, the traditional technologies have not been successful in achieving this. At the same time, the concerns about emerging and re-emerging diseases urge the need to develop technologies that can be rapidly applied to combat the new challenges. Within the last two decades, the research of vaccine technologies has taken several directions to achieve safe, efficient, and economic platforms or technologies for novel vaccines. This review will give a brief overview of the current state of the novel vaccine technologies, new vaccine candidates in clinical trial phases 1–3 (listed by European Medicines Agency (EMA) and Food and Drug Administration (FDA)), and vaccines based on the novel technologies which have already been commercially available (approved by EMA and FDA) with the special reference to pandemic COVID-19 vaccines. Key points • Vaccines of the new generation follow the minimalist strategy. • Some infectious diseases remain a challenge for the vaccine development. • The number of new vaccine candidates in the late phase clinical trials remains low.
... 1997b). In another study, enhancement of antigen-specific Th cell proliferation was seen with TNF-a co-injections (Kim et al. 1998c). In addition, a significant enhancement of the CTL response was observed with the co-administration of TNF-a and IL-15 genes with HIV-l DNA immunogens. ...
Chapter
The skin is the largest as well as the most accessible organ of the human body. It has a unique population of resident antigen-presenting cells (Langerhans cells) and, in addition, a specific subset of circulating T cells homes to the skin. The skin is the target organ for most live attenuated vaccines as well as for the majority of vaccines whose immunogen is based on killed microbes, microbiological extracts or recombinant proteins.
... These cytokines activate the T cell processes of proliferation, cytokine production and survival through the activation of signal transducers and activators of transcription (STAT) 3 and STAT5 proteins [12]. Consistent with these known functions, plasmid DNAs expressing IL-2 and IL-15 enhance vaccine-induced T cell responses [13,14]. Moreover, IL-2 has been approved for clinical use in patients with metastatic renal cell carcinoma and melanoma [15,16] and has been tested with adoptively transferred immune cells for treating patients with melanoma [17]. ...
Article
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Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer.
... 66 Co-delivery of proinflammatory cytokines (IL-1α and TNF-α), Th1 cytokines (IL-2, IL-12, IL15 and IL18) with an HIV immunogen also yielded better results with enhancement in antibody and cellular responses. 65,67 Ishii et al. obtained strong HIV-1 specific cytotoxic T-lymphocyte responses on immunization with DNA and liposomes. 68 Sasaki et al. also observed mucosal immune responses to HIV-1 using QS-21 as an adjuvant, which resulted in the induction of Th1 subsets. ...
... Summary of mucosal adjuvants for DNA vaccine. IL-2, IL-6, IL-7, IL-12, IL-15, GM-CSF, MCP-1, MIP-1α, RANTES T cells stimulation Recruit and activate APCs[192][193][194][195][196][197][198][199][200][201][202][203][204][205][206] Abbreviations: LPS, lipopolysaccharide; MLA, monophosphoryl lipid A; GM-CSF, granulocyte-macrophage colony-stimulating factor; MCP-1, monocyte chemoattractant protein-1; MIP-1α, macrophage inflammatory protein-1α; APCs, antigen presenting cells. ...
Article
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Intranasal delivery of DNA vaccines has become a popular research area recently. It offers some distinguished advantages over parenteral and other routes of vaccine administration. Nasal mucosa as site of vaccine administration can stimulate respiratory mucosal immunity by interacting with the nasopharyngeal-associated lymphoid tissues (NALT). Different kinds of DNA vaccines are investigated to provide protection against respiratory infectious diseases including tuberculosis, coronavirus, influenza and respiratory syncytial virus (RSV) etc. DNA vaccines have several attractive development potential, such as producing cross-protection towards different virus subtypes, enabling the possibility of mass manufacture in a relatively short time and a better safety profile. The biggest obstacle to DNA vaccines is low immunogenicity. One of the approaches to enhance the efficacy of DNA vaccine is to improve DNA delivery efficiency. This review provides insight on the development of intranasal DNA vaccine for respiratory infections, with special attention paid to the strategies to improve the delivery of DNA vaccines using non-viral delivery agents.
... 66 Co-delivery of proinflammatory cytokines (IL-1α and TNF-α), Th1 cytokines (IL-2, IL-12, IL15 and IL18) with an HIV immunogen also yielded better results with enhancement in antibody and cellular responses. 65,67 Ishii et al. obtained strong HIV-1 specific cytotoxic T-lymphocyte responses on immunization with DNA and liposomes. 68 Sasaki et al. also observed mucosal immune responses to HIV-1 using QS-21 as an adjuvant, which resulted in the induction of Th1 subsets. ...
... Moreover, these vaccines are capable of eliciting both antigenspecific humoral and cellular immune responses (Kutzler and Weiner 2008). However, one of the problems associated with DNA vaccines is lower DNA-raised antibodies in comparison with that of protein-raised ones (Kim et al., 1998;Tang et al., 2007;Ferraro et al., 2011). The prime-boost strategy, which is the combination of DNA vaccine priming step followed by boosting with related protein or other vectors each expressing similar antigens, can induce high levels of specific humoral immunity and in some cases can confer protection against infectious agents (Ramshaw and Ramsay 2000;Eo et al., 2001). ...
Article
Considerable advances have been made in developing human papillomaviruses (HPV) prophylactic vaccines based on L1 virus-like particles (VLPs). However, there are limitations in the availability of these vaccines in developing countries, where most cases of cervical cancer occur. In the current study, the prime-boost immunization strategies were studied using a DNA vaccine carrying HPV-16 L1 gene (pcDNA/L1) and insect cell baculovirus-derived HPV-16 L1 VLP. The humoral immunity was evaluated by measuring the specific IgG levels, and the T-cell immune response was assessed by measuring different cytokines such as IFN-γ, TNF-α and IL-10. Results showed that although immunization with pcDNA/L1 alone could induce strong cellular immune responses, higher immunogenicity especially antibody response was achieved in pcDNA/L1 priming-VLP boosting regimen. Therefore, we suggest that prime-boost regimen can be considered as an efficient prophylactic and therapeutic vaccine. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
... Molecular or genetic adjuvants are different from the traditional adjuvants in that they consist of gene-expression constructs encoding immunologically important molecules, such as cytokines, chemokines and co-stimulatory molecules (Iwasaki et al., 1997; Kim et al., 1997, 2000; Sin et al., 1999). Previous reports have shown that co-administration of genetic adjuvant constructs with immunogen constructs can modulate antigen-specific immune responses (Kim et al., 1998, 1999a). ...
Article
Department of Pharmacology, Beijing Institute of Ophthalmology, Beijing 100062, PR ChinaIn this study, the immune-modulatory and vaccine effects of using an interleukin (IL)-18 expressionplasmid as a genetic adjuvant to enhance DNA vaccine-induced immune responses wereinvestigated in a mouse herpes simplex virus 1 (HSV-1) challenge model. BALB/c mice wereimmunized by three intramuscular inoculations ofHSV-1 glycoprotein D(gD) DNAvaccine alone orin combination with a plasmid expressing mature IL-18 peptide. Both the serum IgG2a/IgG1 ratioand T helper 1-type (Th1) cytokines [IL-2 and interferon (IFN)-a] were increased significantly by theco-injection of the IL-18 plasmid compared with the injection of gD DNA alone. However, theproduction of IL-10 was inhibited by IL-18 plasmid co-injection. Furthermore, IL-18 plasmid co-injection efficiently enhanced antigen-specific lymphocyte proliferation and the delayed-typehypersensitivityresponse.WhenmicewerechallengedwithHSV-1atthecornea,co-injectionofIL-18 plasmid with gD DNA vaccine showed significantly better protection, manifested as lowercorneal lesion scores and faster recovery. These experiments indicate that co-injection of an IL-18plasmid with gD DNA vaccine efficiently induces Th1-dominant immune responses and improvesthe protective effect against HSV-1 infection.
... High doses of DNA vaccines are typically required to elicit potent immune responses in mice, and the immunogenicity of DNA vaccines in humans has been marginal to date (5)(6)(7). Strategies to augment the immunogenicity of DNA vaccines are therefore being actively developed (8)(9)(10)(11). However, the mechanism of immune priming and the factors that limit the immunogenicity of DNA vaccines remain poorly characterized. ...
Article
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DCs are critical for priming adaptive immune responses to foreign antigens. However, the utility of harnessing these cells in vivo to optimize the immunogenicity of vaccines has not been fully explored. Here we investigate a novel vaccine approach that involves delivering synergistic signals that both recruit and expand DC populations at the site of antigen production. Intramuscular injection of an unadjuvanted HIV-1 envelope (env) DNA vaccine recruited few DCs to the injection site and elicited low-frequency, env-specific immune responses in mice. Coadministration of plasmids encoding the chemokine macrophage inflammatory protein-1α (MIP-1α) and the DC-specific growth factor fms-like tyrosine kinase 3 ligand with the DNA vaccine resulted in the recruitment, expansion, and activation of large numbers of DCs at the site of inoculation. Consistent with these findings, Coadministration of these plasmid cytokines also markedly augmented DNA vaccine-elicited cellular and humoral immune responses and increased protective efficacy against challenge with recombinant vaccinia virus. These data suggest that the availability of mature DCs at the site of inoculation is a critical rate-limiting factor for DNA vaccine immunogenicity. Synergistic recruitment and expansion of DCs in vivo may prove a practical strategy for overcoming this limitation and potentiating immune responses to vaccines as well as other immunotherapeutic strategies.
... Th1 and Treg cells showed no significant differences in LP of IL-15deficient mice and IL-15 transgenic mice compared to their matched WT mice Earlier studies support the classification of IL-15 as a proinflammatory type-1 cytokine [22,53,54]. Moreover, addition of exogenous IL-15 favored human Th1 T cell differentiation in vitro [22]. ...
Article
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Interleukin (IL)-15 has multiple roles in innate and adaptive immunity, especially regarding CD8+ T cells and natural killer cells. However, the role of IL-15 in regulating differentiation of T helper cell subsets and mononuclear phagocytes (MPs) in different tissues in vivo is unknown. Here we report that IL-15 indirectly regulates Th17 but not other Th subsets in the intestinal lamina propria (LP), apparently through effects on MPs. Th17 cells in the LP were more prevalent in IL-15 KO mice than their wild-type counterparts, and less prevalent in IL-15 transgenic mice than their wild-type littermates, even co-caged. MPs from the LP of these mice were sufficient to mimic the in vivo finding in vitro by skewing of cocultured wild type OVA-specific CD4+ T cells. However, production of IL-15 or lack thereof by these MPs was not sufficient to explain the skewing, as addition or blockade of IL-15 in the cultures had no effect. Rather, a skewing of the relative proportion of CD11b+, CD103+ and double positive LP MP subsets in transgenic and KO could explain the differences in Th17 cells. Thus, IL-15 may influence MP subsets in the gut in a novel way that alters the frequency of LP Th17 cells.
... These studies demonstrated a dramatic increase in specific CTL activity when a gag/pol plasmid or an env plasmid was coadministered with an IL-12 plasmid, as compared with results in animals receiving env or gag/pol plasmids alone. The molecular adjuvant activity of several Th1 cytokines (GM-CSF, IL-2, IL-12, IL-15, and IL-18) was then evaluated in mice in a subsequent study by the Weiner group [64]. This study revealed that the IL-12 plasmid was the best driver of MHC-restricted CD8+ CTL activity. ...
Data
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Trial Protocol HVTN 060. (PDF)
... Costimulatory molecules provide secondary signals for additional activation of T cells for increasing vaccine mediated immune response. For example, costimulatory molecule B7.2 delivered through the vector HIV pCgag/pol enhanced the functioning of cytotoxic T lymphocytes [62]. Similarly, TNF superfamily ligands (TNFSFL) have been shown to improve the efficacy of ALVAC HIV-1 vaccines in a Phase III clinical trial [63]. ...
Article
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HIV/AIDS is a leading cause of mortality and morbidity worldwide. In spite of successful interventions and treatment protocols, an HIV vaccine would be the ultimate prevention and control strategy. Ever since identification of HIV/AIDS, there have been meticulous efforts for vaccine development. The specific aim of this paper is to review recent vaccine efficacy trials and associated advancements and discuss the current challenges and future directions. Recombinant DNA technologies greatly facilitated development of many viral products which were later incorporated into vectors for effective vaccines. Over the years, a number of scientific approaches have gained popularity and include the induction of neutralizing antibodies in late 1980s, induction of CD8 T cell in early 1990s, and combination approaches currently. Scientists have hypothesized that stimulation of right sequences of somatic hypermutations could induce broadly reactive neutralizing antibodies (bnAbs) capable of effective neutralization and viral elimination. Studies have shown that a number of host and viral factors affect these processes. Similarly, eliciting specific CD8 T cells immune responses through DNA vaccines hold future promises. In summary, future studies should focus on the continuous fight between host immune responses and ever-evasive viral factors for effective vaccines.
... Although the use of genetic adjuvants is relatively new compared to traditional adjuvants (such as Alum, which has been utilized since 1926), numerous studies with murine models have confirmed their efficacy. The cytokines IL-2 (Kim et al., 1998), IFN-γ (Chow et al., 1998), GM-CSF (Qing et al., 2010) and IL-15 (Kutzler et al., 2005), when introduced together with a DNA vaccine, stimulate a more enhanced cellular and/or humoral immune response. ...
Article
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The cellular DEAD-box helicase DDX41, which functions as an initial sensor for cytoplasmic DNA, is involved in the activation of type I interferon (IFN-1) immune response in olive flounder. A plasmid encoding DDX41 (pEF-D) was introduced into flounder cells in vitro and in vivo. Immune responses induced by DDX41 were evaluated by relative quantification value (ΔΔCt) method of RT-qPCR using specific IFN-related gene primers. Results in in vitro, transcript levels of IFN-1, IRF-3, ISG-15 and IL-1β were significantly higher in pEF-D- than pEF-A-transfected cells, with 15-, 4-, 10- and 32-fold changes, respectively. In vivo, elevated expression of these genes was observed in the kidney of pEF-D group on days 1 and 3 post-treatment. The viral challenge test revealed higher survival rate in fish treated with pEF-D (67.5%) than controls, PBS- and pEF-A-treated fish (45%). Conclusively DDX41 elicits a robust IFN-mediated immune response, validating its adjuvant property.
... This result may be explained by the following reasons. Previous studies have shown that TNF-a play significant roles in promoting dendritic cells maturation and activating the adaptive immunity after viral infection (Su et al., 2008), and TNF-a was used as efficient adjuvants for viral vaccines, such as bovine herpes virus, human immunodeficiency virus and foot and mouth virus in mammals (Kim et al., 1998;Lewis et al., 1997;Su et al., 2008). However, little study about the adjuvant effects of TNF-a on bacterial vaccines was carried out, which suggested that TNF-a tended to play an immune adjuvant effects on the viral vaccines. ...
... The use o f plasmids expressing cytokine genes have also been investigated in order to improve or manipulate the immune response to DNA vaccines. Plasmids encoding IL-2, IL-12, and IL-18 have been studied (Kim et al, 1998;Billaut-Mulot etal, 2001). ...
Thesis
The use of DNA for vaccination has many potential advantages over conventional vaccines. However, one of the drawbacks associated with this type of vaccination is the apparent lack of immunogenicity of plasmid DNA itself. Traditional aluminium-based adjuvants (alum) have been used to augment the immune response to DNA vaccines, however they have been implicated in allergic responses and the emerging condition macrophagic myofasciitis. The use of zinc oxide as an alternative to alum was investigated for protein antigens and plasmid DNA. An adjuvant effect was observed when zinc oxide was used in combination with other known adjuvants (polymeric microparticles or chitosan) to deliver protein antigen. Prime / boost strategies; priming with plasmid DNA and boosting with protein antigen have been shown to augment cellular and humoral immune responses to many antigens. The coadministration of plasmid DNA and protein antigen as either the priming or booster dose elicited a mixed humoral / cellular response, which was superior to the responses generated by the use of either component alone. Microparticles have been shown to augment the immune response to DNA vaccines, however the plasmid DNA can be degraded within the microparticles as a result of the acidic microclimate generated during polymer degradation. In this study, the co-encapsulation of magnesium hydroxide was observed to increase the stability of plasmid DNA inside the microparticles during polymer degradation. Microencapsulated MUC1 plasmid DNA was investigated for use as a cancer vaccine. Naked DNA has previously been shown to suppress tumour growth when administered intramuscularly. In this study, microencapsulated plasmid DNA delivered subcutaneously led to the greatest suppression of tumour growth. These studies show the initial feasibility of the approaches discussed to deliver plasmid DNA for vaccination and to act as potential adjuvants as an alternative to alum for DNA vaccines.
... Preclinical studies have shown that the immunogenicity of DNA vaccines can be substantially increased by the use of cytokine adjuvants [15][16][17][18][19]. Importantly, an engineered plasmid IL-12 genetic adjuvant has been shown to enhance immunogenicity in humans when delivered using the CELLECTRA ® device [20,21]. ...
Article
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: Background: Recurrent respiratory papillomatosis (RRP) is a rare disorder characterized by the generation of papillomas of the aerodigestive tract, usually associated with human papilloma virus (HPV) subtypes 6, 11. INO-3106 is a DNA plasmid-based immunotherapy targeting E6 and E7 proteins of HPV6, in order to create a robust immune T cell response. Methods: Testing of INO-3016 in animal models confirmed immunogenicity of the DNA-based therapy. A single-site open-label Phase 1 study was initiated for patients with HPV6-positive RRP. Patients were dosed with INO-3106 with or without INO-9012, a DNA plasmid immunotherapy that encodes IL-12, delivered intramuscularly (IM) in combination with electroporation (EP) with the CELLECTRA® device. Patients received an escalating dose of INO-3106, 3 mg once and then 6 mg for three additional doses, each dose three weeks apart, with the third and fourth doses co-administered with INO-9012. The primary objective of the study was to evaluate the safety and tolerability of INO-3106 with and without INO-9012. The secondary objective was to determine cellular immune responses to INO-3106 with and without INO-9012. Exploratory objectives included preliminary clinical efficacy to the therapy. Results: Three patients were enrolled in this study, of which two had RRP. Study therapy was well-tolerated, with no related serious adverse events and all related adverse events (AEs) were low-grade. Injection site pain was the most common related AE reported. Immunogenicity was evidenced by multiple immune assays showing engagement and expansion of an HPV6-specific cellular response, including cytotoxic T cells. Preliminary efficacy was demonstrated in patients with RRP in the form of reduction in need for surgical intervention for papilloma growth. Prior to intervention, both patients required surgical intervention approximately every 180 days. One patient demonstrated a greater than three-fold increase in surgery avoidance (584 days) and the other patient remains completely surgery-free as of the last contact at 915 days, a greater than 5-fold increase in surgery interval. Conclusion: INO-3106 with and without INO-9012 was well tolerated, immunogenic and demonstrated preliminary efficacy in patients with HPV6-associated RRP aerodigestive lesions. Further clinical study is indicated.
... There are instances where cytokines have been used as an immunological adjuvant to increase the efficacy of plasmid DNA vaccine. It has been demonstrated that the magnitude and nature of the immune responses to DNA vaccines can be regulated by co-administration of a broad panel of cytokine genes [96,97]. Among these cytokine genes, IL-12 gene promotes Th1 response and CMI while IL-4 gene favors Th2 cell development and antibodies production. ...
Article
Japanese encephalitis (JE) has emerged as one of the most important form of viral encephalitis, which accounts for an estimated 70,000 cases each year with approximately 10,000 fatalities. The clinical presentations and outcome of the infection is dependent upon both virulence of viral determinants and host immune responses. The causative pathogen of JE is a virus known as Japanese encephalitis virus (JEV), which penetrates into the CNS from blood and triggers rapid humoral and cell-mediated immune response. Humoral response is crucial for the control of dissemination of JEV infection and the cytokines produced by cell-mediated immunity during JEV infections serve as potent immune mediators. Till date, JE is only vaccine preventable and no complete antiviral treatment is available so far. Further, vaccine-mediated prevention also has certain limitations. Therefore, an understanding of the pathogenesis of JEV infection can enable the researchers to presume the depth of treatment regime. This review highlights the importance of understanding of the immune mechanisms that are operated in the host during JEV infection and would be helpful in improving future vaccination strategy against JEV.
... Different cytokines, such as interleukins (IL-2, IL-6, IL-12), chemokines, granulocyte/macrophage colony-stimulating factor (GM-CSF), costimulatory molecules (CD40, CD80, and CD86), and signaling molecules (Interferon regulatory factor -3) have been used as genetic adjuvants with DNA vaccines [39,40,[42][43][44]48,68]. Genes expressing IFN-γ IL-2, IL-12, IL-15, and IL-18 have been used to stimulate Th1 responses [44,45,70], and IL-4, IL-6, IL-10, IL-13, for Th2 stimulation [42,43,[71][72][73]. The inclusion of genes encoding cytokines, like IL-2 or IL-12, as adjuvants for HIV-1 DNA vaccines is known to increase cell mediated immunity (CMI) [74,75]. ...
Article
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DNA vaccines present one of the most cost-effective platforms to develop global vaccines, which have been tested for nearly three decades in preclinical and clinical settings with some success in the clinic. However, one of the major challenges for the development of DNA vaccines is their poor immunogenicity in humans, which has led to refinements in DNA delivery, dosage in prime/boost regimens and the inclusion of adjuvants to enhance their immunogenicity. In this review, we focus on adjuvants that can enhance the immunogenicity of DNA encoded antigens and highlight the development of a novel cytolytic DNA platform encoding a truncated mouse perforin. The application of this innovative DNA technology has considerable potential in the development of effective vaccines.
... A number of groups have shown that this response can be modulated both quantitatively and qualitatively through coimmunization with cytokine-expressing plasmids (Table 1). Specifically, it was demonstrated that coimmunization with Th1-type cytokines can enhance cellular immunity and bias the immune response toward a Th1-type (e.g., IL-12) response, while Th2-type (e.g., IL-4) cytokines can boost Ab responses and promote a Th2-type bias [8,79,80]. In choosing an adjuvant that provides a Th1-or Th2-biased response, it is important to consider which type of response may be more helpful in contributing to protection. ...
Article
The human body has developed an elaborate defense system against microbial pathogens and foreign antigens. However, particular microbes have evolved sophisticated mechanisms to evade immune surveillance, allowing persistence within the human host. In an effort to combat such infections, intensive research has focused on the development of effective prophylactic and therapeutic countermeasures to suppress or clear persistent viral infections. To date, popular therapeutic strategies have included the use of live-attenuated microbes, viral vectors and dendritic-cell vaccines aiming to help suppress or clear infection. In recent years, improved DNA vaccines have now re-emerged as a promising candidate for therapeutic intervention due to the development of advanced optimization and delivery technologies. For instance, genetic optimization of synthetic plasmid constructs and their encoded antigens, in vivo electroporation-mediated vaccine delivery, as well as codelivery with molecular adjuvants have collectively enhanced both transgene expression and the elicitation of vaccine-induced immunity. In addition, the development of potent heterologous prime-boost regimens has also provided significant contributions to DNA vaccine immunogenicity. Herein, the authors will focus on these recent improvements to this synthetic platform in relation to their application in combating persistent virus infection.
Article
As dendritic cells (DCs) play a critical role in priming antigen-specific immune responses, the efficacy of DNA vaccines may be enhanced by targeting the encoded antigen proteins to DCs. In this study, we constructed a DC-targeted DNA vaccine encoding the Hc domain of botulinum neurotoxin serotype A (AHc) fused with scDEC, a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Intramuscular injections of mice with the DC-targeted DNA vaccine (pVAX1-scDEC-AHc) stimulated more DCs to mature than the non-targeted DNA vaccine (pVAX1-SAHc) in the splenocytes. The DC-targeted DNA vaccine could induce more DCs maturation at the site of inoculation. The DC-targeted DNA vaccine induced stronger AHc-specific humoral immune responses, lymphocyte proliferative responses and protective potency against BoNT/A in mice than did pVAX1-SAHc. Moreover, the DC-targeting DNA vaccine provided effective protection after only two inoculations. In summary, these results showed that the DC-targeted fusion DNA vaccine could generate strong immunity, indicating that maturation of DCs induced by pVAX1-scDEC-AHc may be helpful for priming and boosting immune responses. Thus, we propose that the strategy of targeting antigen to DCs in vivo via DEC205 can enhance effectively the potency of DNA vaccines against BoNTs or other pathogens in an animal model.
Chapter
Using plasmids to express protein antigens in vivo has opened many opportunities in the areas of vaccination against infectious diseases, allergy, and cancer immunotherapy. While the initial experiments showing in vivo expression were performed in muscle, similar results have been obtained in the skin. Targeting the skin has allowed DNA vaccines (also known as gene vaccines, genetic vaccines, or DNA immunization) to take advantage of the abundance of antigen-presenting cells in the skin. Although there are several advantages to the use of plasmid DNA vaccination, some disadvantages will be described as well. Most importantly, DNA immunization results in very strong cell mediated immune responses. While immunostimulatory sequences (ISS) present in the plasmid are required, the mechanism by which DNA vaccines induce these potent immune responses are still not fully elucidated. This review will discuss these various aspects of genetic immunization in greater detail.
Article
Chimeric proteins containing antigen linked to cytokines have shown some promise as vaccine candidates but little is known of their mechanism of action, particularly at the level of the antigen- presenting cell. We have investigated this using a chimeric protein in which an immunodominant T cell epitope from influenza hemagglutinin peptide (HA), recognized in the context of I-Ed, was fused to IL-2. Immature murine dendritic cells (DC) derived from bone marrow (BMDC) were used to present the chimeric protein to a T cell hybridoma with TCR specific for the HA peptide (A5 cell line). HA-IL-2 was found to induce significantly higher T cell activation than HA alone. Although the inclusion of IL-2 and HA separately did increase the response of A5 cells compared to HA alone, they were not as effective as the HA-IL-2 chimeric protein. When an antibody known to block IL-2 receptor α chain (CD25) was included, A5 activation was reduced, suggesting a role for the receptor in this process. Expression of CD25 on A5 cells was low during activation, implying that the effect was mediated by CD25 BMDC. Antigen uptake and processing of HA-IL-2 by BMDC was required since fixing BMDC, prior to antigen exposure, greatly reduced their ability to activate A5 cells. The function of CD25 on DC is currently unknown. Our results suggest this receptor may play a role in antigen uptake and subsequent T cell activation by receptor-mediated endocytosis of antigen attached to IL-2. This finding that may have implications for the development of a new generation of vaccines.
Article
IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN- g) and chemokines (RANTES and macrophage inflammatory protein-1 a) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD41 T cells, as determined by in vivo CD41 T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD41 T cell responses that result in
Article
The best hope for the prevention of HIV/AIDS, especially in resource-poor countries, is an effective preventive vaccine. Considerable effort is being made to develop a safe and efficacious vaccine that will elicit broad, protective immunity to HIV. Most of the current vaccine development strategies focus on viral subunit immunogens, which tend to be less than optimally immunogenic and thus require methods to enhance their ability to elicit good neutralizing antibody and vigorous cell-mediated responses. Although generalized adjuvants have long been used to enhance the immune response to vaccines, the use of cytokine adjuvants with specific immune modulatory functions allows the manipulation of the host response to both enhance overall immunogenicity and direct the nature of the response toward a Th1 or Th2 pathway. This review describes the various HIV immunogens and cytokine formulations that are being considered and the state of knowledge about in vitro studies, preclinial and clinical trials of these cytokine-adjuvanted HIV vaccines.
Article
Cytokines are important regulators of the immune response. They influence immune expression, the development of immunologic memory, and regulation of antigen-specific and nonspecific immune activation as well as allergic responses. In a model system in mice, we have studied the effect of plasmids expressing interleukin (IL)-10 or IL-12 on the modulation of antigen-specific responses. Coadministration of IL-12 or IL-10 genes with DNA immunogens directed the antigen-specific immune response toward a T helper (Th1)-type immunity. In addition to the modulation of antigen-specific immune responses, we studied the induction of delayed-type hypersensitivity (DTH) to contact allergens as an in vivo model of the Th1 response. We found that IL-12 and IL-10 gene-containing plasmids, and not the bacterial plasmid alone, upregulate this response. Our cytokine gene delivery technique demonstrates an important level of control of the magnitude and direction of induced immune responses and could be advantageous in a wide variety of immunotherapeutic strategies.
Article
The direct injection of a naked plasmid DNA vaccine encoding a foreign antigen results in plasmid uptake and protein expression leading to the induction of antigen-specific cellular and humoral immune responses. The ability of DNA vaccine-elicited immune responses to protect against viral and bacterial infections, parasites, cancers, and autoimmune diseases has been well documented in numerous animal models. Phase I human clinical trials have shown that experimental DNA vaccines are safe and well tolerated, however, these preliminary studies indicate that measures must be taken to improve vaccine immunogenicity. One approach to improve the immunogenicity of DNA vaccines is through the co-delivery of cytokine expression plasmids as genetic adjuvants. Studies in a variety of animal models clearly demonstrate that plasmid DNA-encoded immunomodulatory cytokines not only alter the magnitude and direction of the DNA vaccine-elicited immune response, but can also improve vaccine efficacy. These studies suggest that the use of immunomodulatory cytokines with plasmid DNA vaccines may allow clinicians to tailor the resulting immune response to more closely resemble the correlates of protection for a given pathogen.
Article
Deoxyribonucleic acid (DNA) vaccination, also known as genetic immunization, is a novel vaccine technology that has been tested in humans for many current infectious diseases, and has been found to be well-tolerated. The approach has been used to induce protective immunity against infectious pathogens, malignancies, as well as prevent the development of autoimmune disorders in animal models. Moreover, DNA vaccines have been tested for clinical use as both prophylactic and therapeutic agents. For these vaccines, plasmid DNA encoding a polypeptide/protein antigen is introduced into a host where it enters host cells and serves as an epigenetic template for the high efficiency translation of antigen. Although DNA immunogens have been shown to stimulate both the cellular and/or humoral arms of the immune system, improving the potency of these vaccines is clearly important. In this regard, several approaches to improve efficacy are currently being tested and will be discussed in this review. One such approach is to improve the DNA plasmid by introducing codon optimization in the DNA plasmid that improves expression and immunogenicity in animals. A second approach aims to manipulate the host immune response by including immunologic molecular adjuvants as part of the vaccine including T cell costimulatory molecules, cytokines, and chemokines. Using immunologic adjuvants, researchers have tailored the immune response to the DNA vaccine toward a particular Th subtype, allowing for the preferential induction of predominantly cell-mediated or humoral response. In particular, interleukin-15 (IL-15) expands CD8 immune responses in the absence of T cell help, while chemokines attract dendritic cells as well as other professional antigen presenting cells, directly activating T and/or B cells in the periphery. Furthermore, priming with such adjuvanted DNAs and boosting with T cell costimulatory molecules further enhances antigen-specific immune response to the DNA antigen. Combining these approaches may be particularly useful against human immunodeficiency virus (HIV) infection, in which both cell-mediated and humoral immune responses are required to fight infection. Ultimately, clinical evidence of these approaches may influence not just how we approach HIV treatment, but also treatments for other infectious diseases, autoimmunity, and cancer.
Article
The potential roles of adhesion molecules in the expansion of T cell-mediated immune responses in the periphery were examined using DNA immunogen constructs as model antigens. We coimmunized cDNA expression cassettes encoding the adhesion molecules intracellular adhesion molecule-1 (ICAM-1), lymphocyte function associated-3 (LFA-3), and vascular cell adhesion molecule-1 (VCAM-1) along with DNA immunogens, and we analyzed the resulting antigen-specific immune responses. We observed that antigen-specific T-cell responses can be enhanced by the coexpression of DNA immunogen and adhesion molecules ICAM-1 and LFA-3. Coexpression of ICAM-1 or LFA-3 molecules along with DNA immunogens resulted in a significant enhancement of T-helper cell proliferative responses. In addition, coimmunization with pCICAM-1 (and more moderately with pCLFA-3) resulted in a dramatic enhancement of CD8-restricted cytotoxic T-lymphocyte responses. Although VCAM-1 and ICAM-1 are similar in size, VCAM-1 coimmunization did not have any measurable effect on cell-mediated responses. These results suggest that ICAM-1 and LFA-3 provide direct T-cell costimulation. These observations are further supported by the finding that coinjection with ICAM-1 dramatically enhanced the level of interferon-gamma (IFN-gamma) and beta-chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and regulated on activation normal T-cell expression and secreted (RANTES) produced by stimulated T cells. Through comparative studies, we observed that ICAM-1/LFA-1 T-cell costimulatory pathways are independent of CD86/CD28 pathways and that they may synergistically expand T-cell responses in vivo.
Article
IL-1β is known as a pro-inflammatory cytokine and plays a pivotal role in regulating immune response. IL-1β has been shown to influence immune responses in Japanese flounder Paralichthys olivaceus. To investigate the immune responses, a plasmid construct of pcDNA3.1-driven Japanese flounder IL-1β (pcDNA3.1-JFIL-1β) was co-injected into the muscle with Bovine serum albumin (BSA), as an antigen model, or pCI-neo driven with GFP (pCI-neo-GFP) as a vaccine model compared with the antigen or vaccine model alone, respectively. The IL-1β expression in the muscle was dramatically elevated in fish injected with pcDNA3.1-JFIL-1β on a day after injection, and the induction level was significantly higher than control groups. Moreover, pcDNA3.1-JFIL-1β significantly stimulated the gene expression of IL-1β in the kidney. The pcDNA3.1-JFIL-1β enhanced the antibody titer against BSA at 30 days after injection. In the DNA vaccine model, the antibody titer against GFP was also higher in the fish injected with pcDNA3.1-JFIL-1β than the group that injected pCI-neo-GFP alone. These results suggest that the pcDNA-driven Japanese flounder IL-1β could have potential immunoadjuvant effects.
Article
Compared with conventional approaches, DNA vaccine technology is a relatively new methodology for producing effective vaccines and has the major advantage of being simple and not requiring any special techniques for the purification and characterisation of recombinant proteins in the correct structure and conformation. The beauty of this approach lies in the ability of DNA vaccines to induce both cellular and humoral responses, thereby having special applications for making improved prophylactic vaccines against diseases for which traditional approaches have failed. Furthermore, the technology also provides an opportunity for developing therapeutic vaccines to treat chronic diseases such as HIV infection and viral hepatitis. These types of vaccines utilise advances in the fields of immunology and molecular biology to tailor more specifically the types of immune responses needed (cellular and/or humoral) against selected targets. In addition, DNA vaccines are potentially safer because, by delivering only the gene(s) encoding the particular immunogen(s) against which a protective or therapeutic immune response is desired, one can avoid the limitations and risks of certain other approaches such as live attenuated pathogens. So far, the efficacy of DNA vaccines has been low to modest in clinical trials, therefore significant efforts have been made to increase the potency of DNA vaccines by increasing the expression levels of the gene of interest, developing novel formulations and delivery technologies, and utilising biological adjuvants. Second-generation DNA vaccines are under development. This review will focus on the status of DNA vaccines against disease and the progress made in increasing the potency and efficacy of DNA vaccines.
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Vaccination is one of the most efficient methods in preventing diseases, and constant innovation in developing new and efficient vaccines is necessary. Adjuvants are components added or conjugated along with the vaccine to enhance the efficiency of the vaccine by improving antigen transport and antigen presentation as well as providing long-lasting immunity, especially in subunit, conjugated, and DNA vaccines. Therefore, adjuvant discovery and formulations are essential parts of vaccine development. Even though inactive and live attenuated vaccines are still widely used and a reliable vaccination approach, polysaccharide-toxoid conjugated vaccines and subunit vaccines also play a pivotal role in eliminating several infectious diseases. A balanced Th1 and Th17 immune response is essential to provide protection against M. tuberculosis infection; that might be achieved through appropriate TLR signaling. Different adjutants can stimulate different inflammatory responses and cytokine production by interacting with various pattern recognition receptors (PRRs) such as NODs, TLRs, RIG-1, etc. Due to the emergence of new diseases, reemerging diseases, and drug-resistant infections such as tuberculosis, the discovery of new vaccine candidates and adjuvants/adjuvant formulations is necessary. CAF01, AS01E, IC31, and GLA‐SE and biomaterial adjuvants in the micro- and nanoscale such as dextran, chitosan, delta inulin, and PLGA are a few notable adjuvants in TB vaccine clinical trials. Nevertheless, a complete understanding of the signaling pathways regarding adjuvants and immune system interaction, biocompatibility, biodegradability, and toxicity of the adjuvants is needed.
Article
Vaccination is the most appropriate way to control pathogens that threaten the global aquaculture industry. Unfortunately, vaccines generally do not provide satisfactory immune protection, especially those based on inactivated pathogens or recombinant antigens. Thus, it is necessary for adjuvants or immunostimulants to be used to improve the vaccine efficacy. Traditional adjuvants such as aluminium salts adjuvant and Freund’s adjuvant, are the most effective known adjuvants in aquaculture; however, due to their disadvantages, such as low adjuvanticity, short lasting time and serious side effects in vivo were also occurred. Searching for some combinations or alternative molecules of them as adjuvants is feasible to increase animal welfare without lowering the levels of immune protection. Recent advances in immunology confirmed that cytokines used as adjuvants have the advantages of eliciting the expression of costimulatory molecular and polarization of antigen‐presenting cells compared with aluminium slats adjuvant and oil adjuvant in fish farming. Moreover, the effects of most adjuvants are exerted mainly through the induction of cytokines production. Nowadays, cytokines have been evaluated for their abilities to offer effective adjuvant activities in both animal models and human studies. In this review, we summarized previous studies performed with different Th cytokines used as adjuvants on different types of fish vaccines, focusing on their protective efficacies and effects on the fish immune system as they delivered in vivo.
Article
Vaccination still remains the most widely used method of controlling infectious diseases in both humans and animals. In view of this, considerable research effort is being devoted to the discovery of new vaccines, and to the refinement of existing ones, with the aim of making them safer, cheaper, and above all, more efficacious. Among the novel developments in vaccine technology is the use of naked DNA encoding the relevant immunogenic component of pathogens, which has been shown to confer protection on animals. Several advantages, such as stability, stimulation of both cellular and humoral immune responses, and antigen expression over prolonged periods, make DNA vaccines an attractive alternative to conventional ones. This paper highlights the progress made so far with the new technology in terms of its immunogenicity, concerns regarding its safety, and future research directions.
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Mycobacterium tuberculosis (Mtb) infection is one of the leading causes of death worldwide. The Modified Vaccinia Ankara (MVA) vaccine vector expressing the mycobacterial antigen 85A (MVA85A) was demonstrated to be safe, although it did not improve BCG efficacy, denoting the need to search for improved tuberculosis vaccines. In this work, we investigated the effect of IL-12 DNA-as an adjuvant-on an Ag85A DNA prime/MVA85A boost vaccination regimen. We evaluated the immune response profile elicited in mice and the protection conferred against intratracheal Mtb H37Rv challenge. We observed that the immunization scheme including DNA-A85A+DNA-IL-12/MVA85A induced a strong IFN-γ production to Ag85A in vitro, with a significant expansion of IFN-γ + CD4 + and IFN-γ + CD8 + anti-Ag85A lymphocytes. Furthermore, we also detected a significant increase in the proportion of specific CD8 + CD107 + T cells against Ag85A. Additionally, inclusion of IL-12 DNA in the DNA-A85A/MVA85A vaccine scheme induced a marked augment in anti-Ag85A IgG levels. Interestingly, after 30 days of infection with Mtb H37Rv, DNA-A85A+DNA-IL-12/MVA85A vaccinated mice displayed a significant reduction in lung bacterial burden. Together, our findings suggest that IL-12 DNA might be useful as a molecular adjuvant in an Ag85A DNA/MVA prime-boost vaccine against Mtb infection.
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The viral cause of AIDS was not established until 1983 (BARRE-SINNOUSSI et al. 1983; GALLO et al. 1983; LEVY et al. 1984), although HIV could have existed in the human population for 50–200 years previously (LI et al. 1988; SMITH et al. 1988; Yokoyama et al. 1988) and may have found its way to Europe and North America as early the 1960s (CORBITT et al. 1990; GARRY et al. 1988; HUMINER et al. 1987). The epidemic continues to sweep across the world, making its most recent inroads in Asia (MANN and TARANTOLA 1996). Vaccines to prevent other viral diseases have been highly successful (PLOTKIN and MORTIMER 1994), and enthusiasm for obtaining one to contain AIDS, once HIV was identified as its cause, was high (RUSSELL 1984). Others pointed out apparently daunting obstacles (ADA 1988; COHN and STEINMAN 1988; FERDINAND et al. 1987; LEVY 1988; NATHANSON and GONZALES-SCARANO 1989), and the intervening years have seen swings of optimism and pessimism about a timetable for eventual success.
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A major advantage of DNA vaccination is the ability to induce both humoral and cellular immune responses. DNA vaccines are currently used in veterinary medicine, but have not achieved widespread acceptance for use in humans due to their low immunogenicity in early clinical studies. However, recent clinical data have re-established the value of DNA vaccines, particularly in priming high-level antigen-specific antibody responses. Several approaches have been investigated for improving DNA vaccine efficacy, including advancements in DNA vaccine vector design, the inclusion of genetically engineered cytokine adjuvants, and novel non-mechanical delivery methods. These strategies have shown promise, resulting in augmented adaptive immune responses in not only mice, but also in large animal models. Here, we review advancements in each of these areas that show promise for increasing the immunogenicity of DNA vaccines.
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Delivery of transgenes has emerged as a useful means of activating immune-based antiviral prophylaxis and therapy. Several studies have now been performed to advance the technology to counter infections caused by human immunodeficiency virus-1, hepatitis B virus (HBV), hepatitis C virus, herpes simplex viruses, and human papillomaviruses. The versatility of methods of manipulating DNA, the scope for inclusion of multiple immunogenic genes in the vectors, and the ability to induce cell-mediated and humoral immune responses are key advantages of the approach. However, inadequate expression of the immunogenic transgenes has hindered efficacy of the technology and optimization of design and delivery of expression cassettes is vital. Delivery of plasmids containing immunogenic transgenes has been improved through use of techniques such as electroporation and administration of transgenes within lipoplex formulations. Recombinant viral vectors, especially those derived from poxviruses and adenoviruses, also improve delivery and have shown utility as vaccines. The prime-boost strategy, which entails sequential administration of different types of vaccination (e.g., DNA electroporation followed by injection with a recombinant virus), augments immunogenicity of transgenes. Vectored immunoprophylaxis also has potential use in antiviral therapy. The technology entails engineering of viral vectors to express virus-neutralizing immunoglobulins or immunoglobulin-like proteins. Engineering T cell receptors and chimeric antigen receptors to recognize viral epitopes may be used to augment immune responses to viruses such as HBV. Although results from studies are promising, further refinements are needed before gene-based immunization is widely used to protect against viruses in a clinical setting.
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DNA immunizadon holds great promise for providing safe and inexpensive vaccines for many infectious pathogens, including HIV-1 (Donnelly, 1997; Kim, 1997a). The direct injection of foreign genes by genetic immunization has resulted in specific immune responses that exhibit characteristics of protective immunity against a number of infectious agents in a variety of animal models. Genetic vaccination cassettes targeting each of HIV-1’s three major genes (env, gag, and pol), regulatory genes, and accessory genes have been developed and studied in small animals, primates, and humans (Donnelly, 1997; Weiner, 1999). Developing successful vaccines for HIV-1 will likely involve targeting multiple antigenic components of the virus to direct and empower the immune system to protect the host from viral infection. Such a collection of immunization cassettes should be capable of stimulating broad immunity against both humoral and cellular epitopes, thus giving a vaccine the maximum ability to deal with viral immune escape. DNA vaccines can be combined with other vaccines including recombinant protein, poxvirus, adenovirus, as well as others to further enhance initial immune responses (Letvin, 1997; Robinson, 1999). It is also important to consider the use of optimized vectors to enhance the level of antigen expression. In addition, the potential of the molecular adjuvant co-administration to dramatically enhance and regulate the antigen-specific humoral and cellular immune responses induced by DNA immunogens represents important new avenue for vaccine and immune therapeutic exploration.
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Nucleic acid vaccination (NAV) is a collection of techniques whose common theme is the introduction of an antigen gene into an animal for the purpose of inducing an immune response to the protein antigen. The chapter discusses the immune pathways used in NAV. This chapter focuses on the immune mechanisms that are involved in the generation of an immune response after NAV and aims to develop a tentative model that outlines the immune pathways involved in generating an immune response for each form of NAV that can be used to predict ways to enhance and modify the immune response. The chapter describes the nomenclature, a short history, and vectors of NAV for further clarification. This study emphasizes that NAV is not one method, but rather consists of a number of techniques. There isn't one best method of nucleic acid vaccination. The investigator can select the procedure that provides the type of immunity that is desired. In the few years since its development, NAV has provided solutions to two long-standing problems in vaccine development. The chapter provides a simple and safe way of inducing cellular immunity without using infectious virus. Also, it has furnished a way to make a vaccine against influenza that protects across 30 years of strain variation.
Article
Gene Therapy for Viral Infections provides a comprehensive review of the broader field of nucleic acid and its use in treating viral infections. The text bridges the gap between basic science and important clinical applications of the technology, providing a systematic, integrated review of the advances in nucleic acid-based antiviral drugs and the potential advantages of new technologies over current treatment options. Coverage begins with the fundamentals, exploring varying topics, including harnessing RNAi to silence viral gene expression, antiviral gene editing, viral gene therapy vectors, and non-viral vectors. Subsequent sections include detailed coverage of the developing use of gene therapy for the treatment of specific infections, the principles of rational design of antivirals, and the hurdles that currently face the further advancement of gene therapy technology. • Provides coverage of gene therapy for a variety of infections, including HBV, HCV, HIV, hemorrhagic fever viruses, and respiratory and other viral infections • Bridges the gap between the basic science and the important medical applications of this technology • Features a broad approach to the topic, including an essential overview and the applications of gene therapy, synthetic RNA, and other antiviral strategies that involve nucleic acid engineering • Presents perspectives on the future use of nucleic acids as a novel class of antiviral drugs • Arms the reader with the cutting-edge information needed to stay abreast of this developing field.
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Trial Protocol HVTN 063. (PDF)
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Any substance (molecule) capable of inducing a specific immune response (humoral or cellular) against that substance, or can be recognized by products of an immune response, such as antibodies or lymphocytes, is commonly called an antigen. Antigens exist in many forms: typically they can be simple molecules, toxins, chemicals, proteins, carbohydrates, lipids or nucleic acids derived from invading microorganisms, such as viruses, bacteria, protozoans, and fungi, or other foreign substances that are usually not found in the body (non-self or heteroantigens). In addition, the body's tissue and cells, including cancer cells, can also have antigens (self or autoantigens, or mutated neoantigens) in them and can cause an immune response (Zamvil et al., 1986; Khodadoust et al., 2017). These autoantigens can be used as biomarkers to identify those specific tissues or cells (Schumacher and Schreiber, 2015; Balachandran et al., 2017). Although antigens exist in numerous forms, they all share a common mechanism of triggering an immune response. The immune system in humans and other mammals can be divided into two general categories: the innate, which is a more general immune system that can recognize foreignness, but not specific antigens, and the adaptive, which is more a specialized immune system with receptors for specific antigens. These two systems work closely together and take on different tasks—while the innate immune response is immediate, the adaptive immune response is not. However, the effect of the adaptive immune response is long-lasting, highly specific, and manifests sustained long-term memory (Janssen et al., 2003; Polonsky et al., 2018).
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Objective: Evaluation of Bax encoding plasmid for increasing efficacy of DNA vaccine plasmid encoding gB of Herpes Simplex Virus type 1. Materials and Methods: We compared three different dosages of Bax encoding plasmid (pcbax) including 10, 25 and 50 μg of plasmid DNA. They were co-injected ineradermally with glycoprotein B (gB) of herpes simplex virus (HSV)-1 encoded plasmid (pcgB) in C57BL/6 mice to elicit immune responses to protect against lethal HSV-1 challenge. Immune responses to the antigen were assessed by lymphocyte proliferative responses and cytokine (INF-γ and IL-4) release assays. Results: The study demonstrates that the mice immunized with 25 μg pcbax together with pcgB have more efficient protection than the mice immunized with 10 and 50 μg of pcbax and pcgB. Analysing of cell-mediated responses show that the mice immunized with 25μg pcbax and pcgB induce stronger lymphocyte proliferative responses and higher levels of INF-γ and IL-4 compared to the mice are received 10 and 50 μg of pcbax and pcgB. Conclusion: The data show that co-immunization with 25 μg of pcbax and pcgB increase immune responses compared to 10 and 50 μg of pcbax and pcgB. This can be considered a promising approach for development an efficient DNA vaccine against HSV-1 or other pathogens.
Thesis
In order to better understand how CD4+ T cells interact in vivo, a mouse model was established, involving the combined adoptive transfer of two populations of T cell receptor (TCR)-transgenic T cells and particle-mediated DNA delivery (PMDD) for the control of antigen presentation by dendritic cells. Since PMDD is a relatively recent method of immunisation, its effect on CD4+ T cells needed to be documented in the context of adoptive transfer models. These preliminary studies allowed the quantification of the CD4+ T cell responses to PMDD in vivo, and in particular, the appearance of specialised activated CD4+ T cells, namely Thl and Th2, and demonstrated the crucial role of cytokines in the early phase of the immune response. Such models should prove useful for testing the concomitant inoculation of DNA antigen and immunomodulators, in the scope of rectifying the balance between these T cell subsets as a strategy for the treatment of many pathological conditions. These models were also used to examine the influence of the genetic background and the dose of DNA administered. A comparative study on migration and activation of na'ive versus polarised CD4+ T cells was also carried out. To examine the cooperation between CD4+ T cells of different specificity, two populations of TCR-transgenic T cells recognising different antigens were co-transferred into the same adoptive host. PMDD was used as a tool for in vivo co-expression of two antigens, either on the same dendritic cell (linked) or not (unlinked). It was found that polarised T cells may considerably influence the fate of naive T cells according to their cytokine profile. Cytokines confined to the dendritic cell cluster microenvironment appeared to play a more important role in the instruction of responding T cells. Moreover, linked presentation of two antigens to clones with a high and a low clonal frequency resulted in a strongly enhanced activation and effector function in the latter. These results demonstrated that cooperation between CD4+ T cells dominates over competition, is more efficient upon linkage with the same antigen-presenting cell and leads to better T cell instruction / deviation.
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In the present study we demonstrate that human monocytes activated by lipopolysaccharides (LPS) were able to produce high levels of interleukin 10 (IL-10), previously designated cytokine synthesis inhibitory factor (CSIF), in a dose dependent fashion. IL-10 was detectable 7 h after activation of the monocytes and maximal levels of IL-10 production were observed after 24-48 h. These kinetics indicated that the production of IL-10 by human monocytes was relatively late as compared to the production of IL-1α, IL-1β, IL-6, IL-8, tumor necrosis factor Oi(TNFα), and granulocyte colony-stimulating factor (G-CSF), which were all secreted at high levels 4-8 h after activation. The production of IL-10 by LPS activated monocytes was, similar to that of IL-1α, IL-1β, IL-6, IL-8, TNFα, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF, inhibited by IL-4. Furthermore we demonstrate here that IL-10, added to monocytes, activated by interferon γ(IFN-γ), LPS, or combinations of LPS and IFN-γ at the onset of the cultures, strongly inhibited the production of IL-1α, IL-1β, IL-6, IL-8, TNFα, GM-CSF, and G-CSF at the transcriptional level. Viral-IL-10, which has similar biological activities on human cells, also inhibited the production of TNFα and GM-CSF by monocytes following LPS activation. Activation of monocytes by LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies resulted in the production of higher amounts of cytokines relative to LPS treatment alone, indicating that endogenously produced IL-10 inhibited the production of IL-1α, IL-1β, IL-6, IL-8, TNFα, GM-CSF, and G-CSF. In addition, IL-10 had autoregulatory effects since it strongly inhibited IL-10 mRNA synthesis in LPS activated monocytes. Furthermore, endogenously produced IL-10 was found to be responsible for the reduction in class II major histocompatibility complex (MHC) expression following activation of monocytes with LPS. Taken together our results indicate that IL-10 has important regulatory effects on immunological and inflammatory responses because of its capacity to downregulate class II MHC expression and to inhibit the production of proinflammatory cytokines by monocytes.
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Tumor necrosis factor-alpha occupies a central role in rheumatoid arthritis (RA) pathogenesis. We now report that interleukin-15 (IL-15) can induce TNF-alpha production in RA through activation of synovial T cells. Peripheral blood (PB) T cells activated by IL-15 induced significant TNF-alpha production by macrophages via a cell-contact-dependent mechanism. Freshly isolated RA synovial T cells possessed similar capability, and in vitro, IL-15 was necessary to maintain this activity. IL-15 also induced direct TNF-alpha production by synovial T cells. In contrast, IL-2 induced significantly lower TNF-alpha production in either cell-contact-dependent or direct culture, and IL-8 and MIP-1 alpha were ineffective. Antibodies against CD69, LFA-1 or ICAM-1 significantly inhibited the ability of T cells to activate macrophages by cell contact.
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Interleukin 10 (IL-10) and viral IL-10 (v-IL-10) strongly reduced antigen-specific proliferation of human T cells and CD4+ T cell clones when monocytes were used as antigen-presenting cells. In contrast, IL-10 and v-IL-10 did not affect the proliferative responses to antigens presented by autologous Epstein-Barr virus-lymphoblastoid cell line (EBV-LCL). Inhibition of antigen-specific T cell responses was associated with downregulation of constitutive, as well as interferon gamma- or IL-4-induced, class II MHC expression on monocytes by IL-10 and v-IL-10, resulting in the reduction in antigen-presenting capacity of these cells. In contrast, IL-10 and v-IL-10 had no effect on class II major histocompatibility complex (MHC) expression on EBV-LCL. The reduced antigen-presenting capacity of monocytes correlated with a decreased capacity to mobilize intracellular Ca2+ in the responder T cell clones. The diminished antigen-presenting capacities of monocytes were not due to inhibitory effects of IL-10 and v-IL-10 on antigen processing, since the proliferative T cell responses to antigenic peptides, which did not require processing, were equally well inhibited. Furthermore, the inhibitory effects of IL-10 and v-IL-10 on antigen-specific proliferative T cell responses could not be neutralized by exogenous IL-2 or IL-4. Although IL-10 and v-IL-10 suppressed IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by monocytes, it was excluded that these cytokines played a role in antigen-specific T cell proliferation, since normal antigen-specific responses were observed in the presence of neutralizing anti-IL-1, -IL-6, and -TNF-alpha mAbs. Furthermore, addition of saturating concentrations of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha to the cultures had no effect on the reduced proliferative T cell responses in the presence of IL-10, or v-IL-10. Collectively, our data indicate that IL-10 and v-IL-10 can completely prevent antigen-specific T cell proliferation by inhibition of the antigen-presenting capacity of monocytes through downregulation of class II MHC antigens on monocytes.
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Abstract Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-lnduced synthesis of IFN-γ and tumor necrosis factor (TNF)-α by human PBMC. However, unlike IL-4, IL-10 Inhibits neither IL-2-lnduced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-lnduced LAK activity. Moreover, IL-4 inhibits IL-2-lnduced IFN-γ synthesis by purified fresh NK cells, while In contrast the inhibitory effect of IL-10 Is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-a synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-lnduced cytokine synthesis and LAK activity are regulated via different mechanisms.
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Selective growth of T lymphocytes occurred when unfractionated normal human bone marrow cells were cultured with conditioned medium obtained from phytohemagglutinin-stimulated normal human lymphocytes (Ly-CM). Cultures of up to 90 percent T cells have been maintained for more than 9 months. The T cells exhibited a strict growth dependence upon Ly-CM and were consistently negative for Epstein-Barr viral information.
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An immunodominant determinant for cytotoxic T lymphocytes (CTLs) exists in the hypervariable portion of human immunodeficiency virus-1 (HIV-1) gp160. Three mouse CTL lines (specific for isolates MN, RF, and IIIB) were examined for recognition of homologous determinants from distinct isolates. Only MN-elicited CTLs showed extensive interisolate cross-reactivity. Residue 325 played a critical role in specificity, with MN-elicited CTLs responding to peptides with an aromatic or cyclic residue and IIIB-induced cells recognizing peptides with an aliphatic residue at this position. CTL populations with broad specificities were generated by restimulation of IIIB-gp160 primed cells with MN-type peptides that have an aliphatic substitution at 325. This represents an approach to synthetic vaccines that can generate broadly cross-reactive CTLs capable of effector function against a wide range of HIV isolates.
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A few cases have been described of antigenic determinants that are broadly presented by multiple class II MHC molecules, especially murine I-E or human DR, in which polymorphism is limited to the beta chain, and the alpha chain is conserved. However, no similar cases have been studied for presentation by class I MHC molecules. Because both domains of the MHC peptide binding site are polymorphic in class I molecules, exploring permissiveness in class I presentation would be of interest, and also such broadly presented antigenic determinants would clearly be useful for vaccine development. We had defined an immunodominant determinant, P18, of the HIV-1 gp160 envelope protein recognized by human and murine CTL. To determine the range of class I MHC molecules that could present this peptide and to determine whether two HIV-1 gp160 Th cell determinants, T1 and HP53, could also be presented by class I MHC molecules, we attempted to generate CTL specific for these three peptides in 10 strains of B10 congenic mice, representing 10 MHC types, and BALB/c mice. P18 was presented by at least four different class I MHC molecules from independent haplotypes (H-2d, p, u, and q to CD8+ CTL. In H-2d and H-2q the presentation was mapped to the D-end class I molecule, and for Dd, a requirement for both the alpha 1 and alpha 2 domains of Dd, not Ld, was found. HP53 was also presented by the same four different class I MHC molecules to CD8+ CTL although at higher concentrations. T1 was presented by class I molecules in three different strains of distinct MHC types (B10.M, H-2f; B10.A, H-2a; and B10, H-2b) to CTL. The CTL specific for P18 and HP53 were shown to be CD8+ and CD4- and to kill targets expressing endogenously synthesized whole gp160 as well as targets pulsed with the corresponding peptide. To compare the site within each peptide presented by the different class I molecules, we used overlapping and substituted peptides and found that the critical regions of each peptide are the similar for all four MHC molecules. Thus, antigenic sites are broadly or permissively presented by class I MHC molecules even without a nonpolymorphic domain as found in DR and I-E, and these sequences may be of broad usefulness in a synthetic vaccine.
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The regulation of Ig class expression has been a controversial area of research. It is well established that T cells, and/or their products, influence which Ig isotype is produced during an immune response. In this study the regulation of Ig secretion of activated human IgM+/A- B cells was examined. Human T cell supernatants induced PWM-activated IgM+/A- B cells to switch to IgA secretion. Purification of the lymphokine mediating this effect involved hydroxylapatite, ion exchange, and gel filtration chromatography. The purified lymphokine could induce switch of IgM+/A- B cells, and it was also capable of inducing proliferation of Staphylococcus aureus Cowan 1 strain (SAC)-activated IgM+/A- B cells. SDS-PAGE and isoelectric focusing indicated the protein mediating this activity had a molecular mass of approximately 14 kDa and a pI of 6.8. These results suggested that the observed activity might be due to low m.w. B cell growth factor (LMW-BCGF), a lymphokine which is capable of inducing proliferation of SAC-activated B cells and has a molecular weight and pI value in the range of the purified protein. Indeed, rLMW-BCGF was able to switch IgM+/A- B-cells to IgA expression and secretion as well as induce the proliferation of SAC-activated IgM+/A- B cells. These results demonstrate that LMW-BCGF is capable of inducing PWM-activated IgM+/A- B-cells to switch to IgA possibly by providing a proliferation signal which induces clonal expansion of IgM+/A- B cells, the progeny of which express a range of isotypes including IgA. This study also demonstrates that lymphokine induced isotype switching involves an intermediate stage of B cell development where human B cells coexpress IgM and a downstream isotype on their surface.
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To produce an immune reaction against a foreign protein usually requires purification of that protein, which is then injected into an animal. The isolation of enough pure protein is time-consuming and sometimes difficult. Here we report that such a response can also be elicited by introducing the gene encoding a protein directly into the skin of mice. This is achieved using a hand-held form of the biolistic system which can propel DNA-coated gold microprojectiles directly into cells in the living animal. Genetic immunization may be time- and labour-saving in producing antibodies and may offer a unique method for vaccination.
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Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-induced synthesis of IFN-gamma and tumor necrosis factor (TNF)-alpha by human PBMC. However, unlike IL-4, IL-10 inhibits neither IL-2-induced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-induced LAK activity. Moreover, IL-4 inhibits IL-2-induced IFN-gamma synthesis by purified fresh NK cells, while in contrast the inhibitory effect of IL-10 is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-alpha synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-induced cytokine synthesis and LAK activity are regulated via different mechanisms.
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A role for antigen-binding B cells in the induction of antigen-dependent B cells producing antigen-nonspecific immunoglobulins (nIFC) is analyzed. An in vitro immune response system was used; sheep red blood cells were employed as the antigen. It is shown that cocultivation of antigen-binding B cells with resting naive splenocytes in the presence of homologous antigen resulted not only in an increase in the number of antibody-forming cells (AFC), but also in a sharp increase in nIFC. Cocultivation of G0 splenocytes with primed B cells depleted for antigen-binding cells did not affect AFC induction, and only slightly augmented the number of nIFC. It is suggested that some of the nIFC appearing after antigenic stimulation arise from resting B lymphocytes in response (direct or indirect) to nonspecific B-cell factor(s).
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IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.
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The biological effects of tumor necrosis factor (TNF) include the enhancement of fibroblast proliferation, the secretion of collagenase and prostaglandin E2 (PGE2) by fibroblasts, and the resorption of bone and cartilage, suggesting a role for this cytokine in arthritic conditions. To investigate this, we measured the levels of TNF in synovial fluids and evaluated its secretion by synovial fluid mononuclear cells and tissues from patients with rheumatoid arthritis, osteoarthritis, and seronegative arthritis and normals. TNF was found to be secreted in all arthritic conditions but not in normals. The levels of TNF were highest in synovial fluid and correlated with interferon-gamma (IFN-gamma) levels but not PGE2. The production of TNF was stable in a single joint for 3 to 6 months. Using immunohistochemical staining, TNF was localized to mononuclear cells in the lining layer, sublining, and perivascular areas of synovial tissue. The secretion of TNF by rheumatoid synovial fluid mononuclear cells was inhibited by PGE2, while IFN-gamma enhanced its production in those cells which were spontaneously secreting TNF. Our data suggest that TNF may play a role in various arthritic diseases.
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A cytokine synthesis inhibitory factor (CSIF) is secreted by Th2 clones in response to Con A or antigen stimulation, but is absent in supernatants from Con A-induced Th1 clones. CSIF can inhibit the production of IL-2, IL-3, lymphotoxin (LT)/TNF, IFN-gamma, and granulocyte-macrophage CSF (GM-CSF) by Th1 cells responding to antigen and APC, but Th2 cytokine synthesis is not significantly affected. Transforming growth factor beta (TGF-beta) also inhibits IFN-gamma production, although less effectively than CSIF, whereas IL-2 and IL-4 partially antagonize the activity of CSIF. CSIF inhibition of cytokine synthesis is not complete, since early cytokine synthesis (before 8 h) is not significantly affected, whereas later synthesis is strongly inhibited. In the presence of CSIF, IFN-gamma mRNA levels are reduced slightly at 8, and strongly at 12 h after stimulation. Inhibition of cytokine expression by CSIF is not due to a general reduction in Th1 cell viability, since actin mRNA levels were not reduced, and proliferation of antigen-stimulated cells in response to IL-2, was unaffected. Biochemical characterization, mAbs, and recombinant or purified cytokines showed that CSIF is distinct from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IFN-gamma, GM-CSF, TGF-beta, TNF, LT, and P40. The potential role of CSIF in crossregulation of Th1 and Th2 responses is discussed.
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Interleukin-2 (IL-2) binds to two distinct receptor molecules, the IL-2 receptor alpha (IL-2R alpha, p55) chain and the newly identified IL-2 receptor beta (IL-2R beta, p70-75) chain. The cDNA encoding the human IL-2R beta chain has now been isolated. The overall primary structure of the IL-2R beta chain shows no apparent homology to other known receptors. Unlike the IL-2R alpha chain, the IL-2R beta chain has a large cytoplasmic region in which a functional domain (or domains) mediating an intracellular signal transduction pathway (or pathways) may be embodied. The cDNA-encoded beta chain binds and internalizes IL-2 when expressed on T lymphoid cells but not fibroblast cells. Furthermore, the cDNA gives rise to the generation of high-affinity IL-2 receptor when co-expressed with the IL-2R alpha chain cDNA.
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During a sublethal murine infection with Listeria monocytogenes cells, tumor necrosis factor (TNF) activity was detectable in neither sera nor spleen homogenates at any stage of the infection when a bioassay with L-929 cells (less than 4 U/ml) was used. However, injecting the mice with an immunoglobulin fraction obtained from a rabbit hyperimmunized with recombinant murine TNF-alpha resulted in acceleration of listeriosis. When 1 mg of anti-TNF antibody was injected per mouse, all the mice died from listeriosis, even though the infectious dose was sublethal for the untreated controls. The antigen-specific elimination of the bacterium from the spleens and livers of anti-TNF antibody-treated mice was delayed, depending on the dose of the antibody injected. Endogenous TNF seemed to be produced early in infection, because suppression of antilisterial resistance was significant when a single injection of anti-TNF antibody was given between day zero and day 2 of infection. The effect of endogenous TNF on antilisterial resistance was due to neither regulation of alpha interferon (IFN-alpha) and IFN-gamma production nor induction of IFN-beta subtype 1 (IFN-beta 1), because anti-TNF antibody treated-mice produced normal levels of IFN-alpha and IFN-gamma in the bloodstream during infection and administration of monoclonal anti-murine IFN-beta 1 antibody had no effect on the development of listeriosis. Alternatively, the listericidal activity of peritoneal macrophages of L. monocytogenes-infected mice could be abrogated by injection of anti-TNF antibody in vivo. These results suggest that the lower level of TNF is produced endogenously in mice that received L. monocytogenes infection and that it plays an essential role in the host defense against L. monocytogenes infection.
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MOUSE lymphocytes can be induced in vitro by treatment with phytohaemagglutinin (PHA), or with rabbit anti-mouse serum, and by the mixed lymphocyte reaction, to produce material(s) which is toxic to L cells1. The term lymphotoxins (previously termed lymphocyte cytotoxic factor) is applied here to the toxic factor(s). We report that lymphocytes from several animal species, including man, can be stimulated with phytohaemagglutinin in vitro to release lymphotoxin-like materials.
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The role of factors released by monocytes (M phi) in the activation of human B lymphocytes was examined by studying the effect of an antiserum against human leukocytic pyrogen (LP) on mitogen-stimulated B cell proliferation and the generation of immunoglobulin-secreting cells (ISC) by peripheral blood mononuclear cells (PBM). Antiserum against LP was obtained from rabbits immunized with LP-containing human M phi supernatants. The globulin fraction of this antiserum inhibited pokeweed mitogen- (PWM) stimulated B cell proliferation and the generation of ISC in a concentration-dependent manner, with 50% inhibition of responsiveness observed with 10 micrograms/ml. By contrast, PWM-induced T cell [3H]thymidine incorporation was not inhibited by concentrations of anti-LP as great as 2000 micrograms/ml. The F(ab')2 fraction of anti-LP also inhibited the generation of ISC in response to both PWM and formalinized Staphylococcus aureus, but required 50 micrograms/ml to achieve 50% inhibition. Anti-LP inhibited the generation of ISC only if present during the first 24 hr of a 6 to 7-day incubation; later addition was not inhibitory. Inhibition was more marked in cultures partially depleted of M phi than in whole PBM cultures. Whereas absorption of the anti-LP with PBM failed to remove the capacity to inhibit the generation of ISC, anti-LP-mediated inhibition of responsiveness could be reversed by the addition of crude M phi culture supernatants or a variety of highly purified interleukin 1 (IL 1) preparations, but not by T cell supernatants. These results indicate anti-LP inhibits human B cell activation by removing the requisite M phi-derived factor IL 1 and also confirm that IL 1 plays an essential role in B cell proliferation and the generation of ISC in man.
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By using as an experimental system the induction of growth of a cloned, antigen:Ia-reactive helper T cell line by an antigen receptor-specific monoclonal antibody, we demonstrated that growth requires two essential co-factors, exogenously produced IL 1 and endogenously produced IL 2. The primary role of the IL 1 is in the expression of receptors on the T cell surface for IL 2, rather than for promoting the synthesis of IL 2. The use of a clone-specific activating monoclonal antibody at nanogram amounts to activate a cloned helper T cell should allow a detailed characterization of T cell activation via antigen receptor cross-linking.
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The mechanism underlying the differentiation of CD4+ T cells into functionally distinct subsets (Th1 and Th2) is incompletely understood, and hitherto unidentified cytokines may be required for the functional maturation of these cells. Here we report the cloning of a recently identified IFN-gamma-inducing factor (IGIF) that augments natural killer (NK) activity in spleen cells. The gene encodes a precursor protein of 192 amino acids and a mature protein of 157 amino acids, which have no obvious similarities to any peptide in the databases. Messenger RNAs for IGIF and interleukin-12 (IL-12) are readily detected in Kupffer cells and activated macrophages. Recombinant IGIF induces IFN-gamma more potently than does IL-12, apparently through a separate pathway. Administration of anti-IGIF antibodies prevents liver damage in mice inoculated with Propionibacterium acnes and challenged with lipopolysaccharide, which induces toxic shock. IGIF may be involved in the development of Th1 cells and also in mechanisms of tissue injury in inflammatory reactions.
Article
IL-10 has been shown to be capable of down-regulating several aspects of macrophage function. This study was undertaken to define the association between IL-10 and HIV-1 infection in human macrophages. Infection of macrophages with a monocytotropic strain of the human immunodeficiency virus, HIV-BaL, resulted in expression of IL-10 mRNA within 3 to 12 h after infection, as determined by the reverse transcriptase PCR. Biologically active IL-10 was detected in supernatants from HIV-1-infected macrophages as early as 12 h post-infection. The addition of human rIL-10 to HIV-1-infected macrophage cultures resulted in a significant decrease in the viral replication. In addition, exogenous IL-10 blocked the ability of TNF-alpha to elevate viral replication. To determine whether IL-10 was associated with in vivo infection, lymph nodes from AIDS patients were examined for the presence of IL-10 mRNA by using PCR. IL-10 mRNA was evident in all lymph node tissue examined, but was absent in normal lymph node biopsies. These in vitro and in vivo findings demonstrate a strong and heterogeneous association between HIV-1 infection and IL-10.
Article
Interleukin-12 (IL-12) is a heterodimeric cytokine produced mostly by phagocytic cells in response to bacteria, bacterial products, and intracellular parasites, and to some degree by B lymphocytes. IL-12 induces cytokine production, primarily of IFN-gamma, from NK and T cells, acts as a growth factor for activated NK and T cells, enhances the cytotoxic activity of NK cells, and favors cytotoxic T lymphocyte generation. In vivo IL-12 acts primarily at three stages during the innate resistance/adaptive immune response to infection: 1. Early in the infection, IL-12 is produced and induces production from NK and T cells of IFN-gamma, which contributes to phagocytic cell activation and inflammation; 2. IL-12 and IL-12-induced IFN-gamma favor Th1 cell differentiation by priming CD4+ T cells for high IFN-gamma production; and 3. IL-12 contributes to optimal IFN-gamma production and to proliferation of differentiated Th1 cells in response to antigen. The early preference expressed in the immune response depends on the balance between IL-12, which favors Th1 responses, and IL-4, which favors Th2 responses. Thus, IL-12 represents a functional bridge between the early nonspecific innate resistance and the subsequent antigen-specific adaptive immunity.
Article
This review has summarized information published over the last 5 years on the presence and pathophysiologic role of IL-1 and TNF alpha in RA. The evidence to date shows that 5 of 6 criteria for identifying mediators of tissue damage in human autoimmune diseases are satisfied (Table 1). The last criterion, prevention of clinical progression in patients with RA, is currently being evaluated. Many new therapeutic approaches are currently being developed, including the use of soluble receptors to IL-1 or TNF, monoclonal antibodies to TNF alpha, a specific IL-1 receptor antagonist, and gene therapy with the latter molecule. It should be emphasized that both IL-1 and TNF alpha play important roles in normal host defense; the possible complications of blocking their production or effects need to be carefully evaluated in long-term studies. A recent review has emphasized that although IL-1 and TNF alpha have many overlapping biologic properties, each may exhibit distinct effects in joint disease (99). Anti-TNF treatment may be primarily antiinflammatory but blocking IL-1 may be more effective in preventing cartilage destruction (100). The possibility exists that simultaneous inhibition of TNF alpha and IL-1 may be more therapeutically efficacious than blockade of either agent alone, as was recently demonstrated with IL-1ra and soluble TNF receptors in bacterial cell wall-induced arthritis in rats (101). The next level of clinical studies in rheumatoid arthritis should include the use of two biologic response modifiers together, or one agent combined with a more traditional form of therapy.
Article
Inoculation of plasmid vectors encoding a viral protein into muscle tissue was shown to result in expression of the transantigen and, consequently, an antiviral immune response. Here, we show that coinoculation of a plasmid expressing the glycoprotein of rabies virus with plasmids encoding mouse cytokines modulated the immune response to the viral protein. Coinoculation with a vector expressing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the B and T helper cell activity to rabies virus, while coinoculation with a plasmid expressing interferon-gamma (IFN gamma) resulted in a decrease of the immune response to the viral antigen.
Article
Naive CD4+ T cells when stimulated produce IL-2 as their major lymphokine. Upon priming, these cells develop into cells that produce either IFN gamma, TNF beta, and IL-2 or IL-4 and its congeners. The former cells are designated TH1-like, and the latter TH2-like. Here we review the regulation of the differentiation of naive CD4 cells into IFN gamma- or IL-4-producers. The dominant factors that determine such differentiation are lymphokines and other cytokines. IL-2 itself appears to be required for naive cells to develop into TH1- or TH2-like cells but is not deterministic of their differentiation fate. If IL-4 is also present during the priming period, the resultant CD4+ T cells produce IL-4 upon restimulation; the development of IFN gamma-producing cells is strikingly inhibited by IL-4. In the absence of IL-4, priming for IFN gamma-production occurs, but this is markedly enhanced by IL-12. The role of IFN gamma in enhancing priming for IFN gamma-production is not fully resolved. In some in vitro systems, it appears to act together with IL-12 to enhance such production. Anti-IFN gamma diminishes priming for IFN gamma production in vivo. Lymphokines also exert a "cross-regulatory" or inhibitory effect. As noted above, IL-4 strikingly diminishes priming for IFN gamma production, although this inhibitory effect is blunted in the presence of IL-12. IFN gamma similarly diminishes priming for IL-4 production; this effect is principally observed when low concentrations of IL-4 are used in the priming culture. Although other factors may play a role in the determination of lymphokine-producing phenotype, such as antigen dose, type of antigen-presenting cell, and expression of accessory molecules and hormones, these effects appear to be secondary to the dominant role of the lymphokines and cytokines.
Article
A cytokine was identified that stimulated the proliferation of T lymphocytes, and a complementary DNA clone encoding this new T cell growth factor was isolated. The cytokine, designated interleukin-15 (IL-15), is produced by a wide variety of cells and tissues and shares many biological properties with IL-2. Monoclonal antibodies to the beta chain of the IL-2 receptor inhibited the biological activity of IL-15, and IL-15 competed for binding with IL-2, indicating that IL-15 uses components of the IL-2 receptor.
Article
In early phases of human T-cell lymphotrophic virus I-induced adult T-cell leukemia (ATL), the malignant cell proliferation is associated with an autocrine process involving coordinate expression of interleukin (IL) 2 and its receptor. However, during late-phase ATL, leukemic cells no longer produce IL-2 yet continue to express high-affinity IL-2 receptors. During studies to define pathogenic mechanisms that underlie this IL-2-independent proliferation, we demonstrated that the ATL cell line HuT-102 secretes a lymphokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Conditioned medium from HuT-102, when added to the IL-2-dependent CTLL-2 line, yielded a stimulation index of 230. Since CTLL-2 was purported to be IL-2-specific, we performed a number of studies to exclude IL-2 production by HuT-102. Stim