Sarah Godbehere

Adenovirus Vector-Induced Innate Inflammatory Mediators, MAPK Signaling, As Well As Adaptive Immune Responses Are Dependent upon Both TLR2 and TLR9 In Vivo

Adenovirus (Ad) vectors are promising candidates for both gene transfer and vaccine applications. In this study, we investigated the role of TLR2 in innate and adaptive immune responses to Ad and/or the transgene it expresses following systemic injection. We found that Ad directly activates ERK1/2 in vivo, but that initiation of ERK1/2 activation is primarily a MyD88/TLR2-independent, but Kupffer cell-dependent, event. The complexity of Ad-induced innate immune responses was confirmed when we also found that both TLR2 and MyD88 functions are required for the sustained activation of ERK1/2. Although we found that the initial activation of NF-κB by Ads is dependent upon MyD88, but independent of TLR2 in (non-Kupffer cells) the liver, TLR2 significantly influenced the Ad-induced late phase NF-κB activation. These very rapid responses were positively correlated with subsequent innate immune responses to the Ad vector, as our results confirmed that the induction of several cytokines and chemokines, and the expression of innate immune response genes following Ad injection were TLR2 dependent in vivo. The requirement of TLR2 in Ad-induced innate responses also correlated with significantly altered adaptive immune responses. For example, our results demonstrate that the generation of Ad-neutralizing Abs, and anti-transgene-specific Abs elicited subsequent to Ad vector treatments, are both dependent upon TLR2 functionality. Finally, we found that several Ad-induced innate immune responses are dependent on both TLR2 and TLR9. Therefore, this study confirms that several (but not all) Ad-induced innate and adaptive immune responses are TLR dependent.

Transient Pretreatment With Glucocorticoid Ablates Innate Toxicity of Systemically Delivered Adenoviral Vectors Without Reducing Efficacy

More than 300 human clinical trials utilize recombinant adenoviruses (rAds) as a gene transfer vector, confirming that rAds continue to be of high clinical interest. A primary weakness of rAds is their known propensity to trigger an innate, proinflammatory immune response rapidly after high-dose, systemic administration. In this study, we investigated what affects that pre-emptive treatment with anti-inflammatory glucocorticoids might have upon Ad vector-triggered inflammatory immune responses. We found that a simple pretreatment regimen with Dexamethasone (DEX) can significantly reduce most Ad-induced innate immune responses. DEX prevented rAd induction of systemic cytokine/chemokine releases in a dose-dependent fashion, with higher dosages preventing rAd induction of acute thrombocytopenia, endothelial cell activation, proinflammatory gene induction, and leukocyte infiltration into transduced organs. Transient glucocorticoid pretreatment also significantly reduced rAd-induced adaptive immune responses, including a decreased induction of Ad-neutralizing antibodies (NAbs). Importantly, use of DEX did not reduce the efficacy of rAd-mediated gene transduction nor rAd-derived transgene expression. Our results demonstrate that a simple, pre-emptive and transient glucocorticoid pretreatment is a viable approach to reduce rAd-associated acute toxicities that currently limit the use of Ad vectors in systemic clinical applications.

Complex interactions with several arms of the complement system dictate innate and humoral immunity to adenoviral vectors

The complement system is known to play critical roles in pathogen identification, initiation of innate immune responses and facilitation of adaptive immune responses. Several studies have suggested that recombinant adenoviruses (rAds) interact with proteins of the complement system within minutes of administration. In this study, we assessed the roles of the alternative (Factor B), classical (C1q and C4) and common (C3) arms of the complement system in the innate and humoral response to systemic rAd administration using mice genetically deficient for each of these functions. Although most plasma cytokines and chemokines induced by Ads appeared to be elicited in a C3-dependent manner, we found that rAd-induced thrombocytopenia was dependent on Factor B and C3, implicating the alternative pathway as responsible for this response. Alteration of the complement-dependent transcriptome response after rAd-induced liver gene expression was also found to be Factor B- and C3-dependent. Ad interactions with the classical and alternative arms of the complement system can also be redundant, as many complement-dependent, Ad-induced innate immune responses appeared to be primarily C3-dependent. We also identified a C3 dependence of Ad-mediated induction of the nuclear factor-κB (NF-κB) activation pathway. Finally, we confirmed that humoral immune responses to the vector capsid, and the transgene it encodes, are also complement-dependent.

Sublingual Administration of an Adenovirus Serotype 5 (Ad5)-Based Vaccine Confirms Toll-Like Receptor Agonist Activity in the Oral Cavity and Elicits Improved Mucosal and Systemic Cell-Mediated Responses against HIV Antigens despite Preexisting Ad5 Immunity

ABSTRACT HIV/AIDS continue to devastate populations worldwide. Recent studies suggest that vaccines that induce beneficial immune responses in the mucosal compartment may improve the efficacy of HIV vaccines. Adenovirus serotype 5 (Ad5)-based vectors remain a promising platform for the development of effective vaccines. In an effort to improve the efficacy of Ad5-based vaccines, even in the presence of preexisting Ad5 immunity, we evaluated the potential for an Ad5-based HIV vaccine to induce antigen-specific immune responses following sublingual (s.l.) administration, a route not previously tested in regard to Ad-based vaccines. s.l. vaccination with an Ad5-based HIV-Gag vaccine resulted in a significant induction of Gag-specific cytotoxic T-lymphocyte (CTL) responses in both the systemic and the mucosal compartment. We also show that s.l. immunization not only avoided preexisting Ad5 immunity but also elicited a broad repertoire of antigen-specific CTL clones. Additionally, we confirm for the first time that oral delivery of a vaccine expressing a potent Toll-like receptor (TLR) agonist can stimulate innate immune responses through induction of cytokines and chemokines and activation of NK cells, NKT cells, and macrophages in vivo. These results positively correlated with improved antigen-specific CTL responses. These results could be achieved both in Ad5-naïve mice and in mice with preexisting immunity to Ad5. The simplicity of the s.l. vaccination regimen coupled with augmentation of TLR-dependent pathways active in the oral cavity makes s.l. delivery a promising method for HIV vaccine development specifically, as well as for many other vaccine applications in general.

Adenovirus-based vaccination against Clostridium difficile toxin A allows for rapid humoral immunity and complete protection from toxin A lethal challenge in mice.

Clostridium difficile associated diarrhea (CDAD) is a critical public health problem worldwide with over 300,000 cases every year in the United States alone. Clearly, a potent vaccine preventing the morbidity and mortality caused by this detrimental pathogen is urgently required. However, vaccine efforts to combat C. difficile infections have been limited both in scope as well as to efficacy, as such there is not a vaccine approved for use against C. difficile to date. In this study, we have used a highly potent Adenovirus (Ad) based platform to create a vaccine against C. difficile. The Ad-based vaccine was able to generate rapid and robust humoral as well as cellular (T-cell) immune responses in mice that correlated with provision of 100% protection from lethal challenge with C. difficile toxin A. Most relevant to the clinical utility of this vaccine formulation was our result that toxin A specific IgGs were readily detected in plasma of Ad immunized mice as early as 3 days post vaccination. In addition, we found that several major immuno-dominant T cell epitopes were identified in toxin A, suggesting that the role of the cellular arm in protection from C. difficile infections may be more significant than previously appreciated. Therefore, our studies confirm that an Adenovirus based-C. difficile vaccine could be a promising candidate for prophylactic vaccination both for use in high risk patients and in high-risk environments.

A New Adenovirus Based Vaccine Vector Expressing an Eimeria tenella Derived TLR Agonist Improves Cellular Immune Responses to an Antigenic Target

BACKGROUND Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals. METHODOLOGY/PRINCIPAL FINDINGS In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8(+) and CD8(-) T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions. CONCLUSION/SIGNIFICANCE The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.

Expression of the SLAM Family of Receptors Adapter EAT-2 as a Novel Strategy for Enhancing Beneficial Immune Responses to Vaccine Antigens

Recent studies have shown that activation of the signaling lymphocytic activation molecule (SLAM) family of receptors plays an important role in several aspects of immune regulation. However, translation of this knowledge into a useful clinical application has not been undertaken. One important area where SLAM-mediated immune regulation may have keen importance is in the field of vaccinology. Because SLAM signaling plays such a critical role in the innate and adaptive immunity, we endeavored to develop a strategy to improve the efficacy of vaccines by incorporation of proteins known to be important in SLAM-mediated signaling. In this study, we hypothesized that coexpression of the SLAM adapter EWS-FLI1–activated transcript 2 (EAT-2) along with a pathogen-derived Ag would facilitate induction of beneficial innate immune responses, resulting in improved induction of Ag-specific adaptive immune responses. To test this hypothesis, we used rAd5 vector-based vaccines expressing murine EAT-2, or the HIV-1–derived Ag Gag. Compared with appropriate controls, rAd5 vectors expressing EAT-2 facilitated bystander activation of NK, NKT, B, and T cells early after their administration into animals. EAT-2 overexpression also augments the expression of APC (macrophages and dendritic cells) surface markers. Indeed, this multitiered activation of the innate immune system by vaccine-mediated EAT-2 expression enhanced the induction of Ag-specific cellular immune responses. Because both mice and humans express highly conserved EAT-2 adapters, our results suggest that human vaccination strategies that specifically facilitate SLAM signaling may improve vaccine potency when targeting HIV Ags specifically, as well as numerous other vaccine targets in general.

CR1/2 is an important suppressor of Adenovirus-induced innate immune responses and is required for induction of neutralizing antibodies.

Human complement receptors 1 and 2 are well described as important regulators of innate and adaptive immune responses, having pivotal roles in regulating complement activation (CR1) and B-cell maturation/survival. In contrast, the role of the murine homologs of CR1 and CR2 (mCR1/2) have been primarily defined as modulating activation of the adaptive immune system, with very little evidence available about the role of mCR1/2 in regulating the innate immune responses to pathogens. In this paper, we confirm that mCR1/2 plays an important role in regulating both the innate and adaptive immune responses noted after Adenovirus (Ad)-mediated gene transfer. Our results uncovered a novel role of mCR1/2 in downregulating several complement-dependent innate immune responses. We also unveiled the mechanism underlying the complement-dependent induction of neutralizing antibodies to Ad capsids as a CR1/2-dependent phenomenon that correlates with B-cell activation. These results confirm that Ad interactions with the complement system are pivotal in understanding how to maximize the safety or potency of Ad-mediated gene transfer for both gene therapy and vaccine applications.

TRIF, and TRIF-Interacting TLRs Differentially Modulate Several Adenovirus Vector-Induced Immune Responses

The use of Adenovirus (Ad)-based vectors has proven to be a useful platform for the development of gene therapy and vaccine protocols. The immunological mechanisms underlying these properties need to be identified and understood to foster safer, more efficacious use of this important gene transfer platform. Our recent studies have confirmed an important role for MyD88 dependent toll-like receptor (TLR) pathways as mediators of these responses. In this study, we confirm that TLR3, TLR4 and TRIF (TIR-domain-containing adapter-inducing interferon-β) can also have augmentative or inhibitory roles during Ad-induced immune responses. Importantly, our studies revealed that TLR4 acts to suppress several aspects of the Ad-induced innate immune response, a finding not previously reported for this TLR in any model system. In addition, using MyD88 and TRIF double knockout mice, we demonstrate that the MyD88 and TRIF adaptor proteins can play either additive or redundant roles in mediating certain aspects of Ad vector-induced innate and adaptive immune responses. Furthering this complexity, our model system strongly suggests that non-TLR based systems must not only exist, but also have a significant role to play during Ad vector-mediated induction of adaptive immune responses.

β-Arrestins modulate Adenovirus-vector-induced innate immune responses: Differential regulation by β-arrestin-1 and β-arrestin-2

Adenovirus (Ad)-based vectors have been utilized in human gene transfer clinical trials since 1993. Unfortunately, innate immune responses directed against the Ad capsid and/or its genetic cargo can significantly limit the usage of Ad vectors. Previous studies have demonstrated that several signaling pathways are triggered by Ads, inclusive of TLR-dependent pathways. The G-protein-coupled receptor adaptors beta-arrestin-1 (beta-Arr1) and beta-arrestin-2 (beta-Arr2) are known to have pivotal roles in regulating TLR4 triggered signaling and inflammatory responses. In this study, we examined the role of beta-arrestins in Ad5-vector-induced inflammatory responses. Our studies reveal that both beta-arrestins are capable of modulating Ad5-vector-induced inflammatory responses in vivo and in vitro. Importantly, our studies divulge another level of complexity to these responses, as our results demonstrate beta-Arr1 to be a positive regulator, and beta-Arr2 a negative regulator of Ad5 induced innate immune responses. These data may allow gene therapy biologists to more accurately study the mechanisms underlying Ad5-vector-induced immune responses, and may also direct future efforts to modulate these mechanisms to improve the safety and/or efficacy of this important gene transfer vector.