Maria J. Chiuchiolo

Profound early control of highly pathogenic SIV by an effector memory T-cell vaccine

The acquired immunodeficiency syndrome (AIDS)-causing lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) effectively evade host immunity and, once established, infections with these viruses are only rarely controlled by immunological mechanisms. However, the initial establishment of infection in the first few days after mucosal exposure, before viral dissemination and massive replication, may be more vulnerable to immune control. Here we report that SIV vaccines that include rhesus cytomegalovirus (RhCMV) vectors establish indefinitely persistent, high-frequency, SIV-specific effector memory T-cell (TEM) responses at potential sites of SIV replication in rhesus macaques and stringently control highly pathogenic SIVMAC239 infection early after mucosal challenge. Thirteen of twenty-four rhesus macaques receiving either RhCMV vectors alone or RhCMV vectors followed by adenovirus 5 (Ad5) vectors (versus 0 of 9 DNA/Ad5-vaccinated rhesus macaques) manifested early complete control of SIV (undetectable plasma virus), and in twelve of these thirteen animals we observed long-term (≥1 year) protection. This was characterized by: occasional blips of plasma viraemia that ultimately waned; predominantly undetectable cell-associated viral load in blood and lymph node mononuclear cells; no depletion of effector-site CD4+ memory T cells; no induction or boosting of SIV Env-specific antibodies; and induction and then loss of T-cell responses to an SIV protein (Vif) not included in the RhCMV vectors. Protection correlated with the magnitude of the peak SIV-specific CD8+ T-cell responses in the vaccine phase, and occurred without anamnestic T-cell responses. Remarkably, long-term RhCMV vector-associated SIV control was insensitive to either CD8+ or CD4+ lymphocyte depletion and, at necropsy, cell-associated SIV was only occasionally measurable at the limit of detection with ultrasensitive assays, observations that indicate the possibility of eventual viral clearance. Thus, persistent vectors such as CMV and their associated TEM responses might significantly contribute to an efficacious HIV/AIDS vaccine.

Enhanced Control of Pathogenic Simian Immunodeficiency Virus SIVmac239 Replication in Macaques Immunized with an Interleukin-12 Plasmid and a DNA Prime-Viral Vector Boost Vaccine Regimen

ABSTRACT DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent “blips” in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.

Protective immunity against respiratory tract challenge with Yersinia pestis in mice immunized with an adenovirus-based vaccine vector expressing V antigen.

Abstract The aerosol form of the bacterium Yersinia pestis causes the pneumonic plague, a rapidly fatal disease. At present, no plague vaccines are available for use in the United States. One candidate for the development of a subunit vaccine is the Y. pestis virulence (V) antigen, a protein that mediates the function of the Yersinia outer protein virulence factors and suppresses inflammatory responses in the host. On the basis of the knowledge that adenovirus (Ad) gene-transfer vectors act as adjuvants in eliciting host immunity against the transgene they carry, we tested the hypothesis that a single administration of a replication-defective Ad gene-transfer vector encoding the Y. pestis V antigen (AdsecV) could stimulate strong protective immune responses without a requirement for repeat administration. AdsecV elicited specific T cell responses and high IgG titers in serum within 2 weeks after a single intramuscular immunization. Importantly, the mice were protected from a lethal intranasal challenge of Y. pestis CO92 from 4 weeks up to 6 months after immunization with a single intramuscular dose of AdsecV. These observations suggest that an Ad gene-transfer vector expressing V antigen is a candidate for development of an effective anti-plague vaccine

Protective Immunity Against a Lethal Respiratory Yersinia pestis Challenge Induced by V Antigen or the F1 Capsular Antigen Incorporated into Adenovirus Capsid

The aerosol form of the bacterium Yersinia pestis causes pneumonic plague, a rapidly fatal disease that is a biothreat if deliberately released. At present, no plague vaccines are available for use in the United States, but subunit vaccines based on the Y. pestis V antigen and F1 capsular protein show promise when administered with adjuvants. In the context that adenovirus (Ad) gene transfer vectors have a strong adjuvant potential related to the ability to directly infect dendritic cells, we hypothesized that modification of the Ad5 capsid to display either the Y. pestis V antigen or the F1 capsular antigen on the virion surface would elicit high V antigen- or F1-specific antibody titers, permit boosting with the same Ad serotype, and provide better protection against a lethal Y. pestis challenge than immunization with equivalent amounts of V or F1 recombinant protein plus conventional adjuvant. We constructed AdYFP-pIX/V and AdLacZ-pIX/F1, E1(-), E3(-) serotype 5 Ad gene transfer vectors containing a fusion of the sequence for either the Y. pestis V antigen or the F1 capsular antigen to the carboxy-terminal sequence of pIX, a capsid protein that can accommodate the entire V antigen (37 kDa) or F1 protein (15 kDa) without disturbing Ad function. Immunization with AdYFP-pIX/V followed by a single repeat administration of the same vector at the same dose resulted in significantly better protection of immunized animals compared with immunization with a molar equivalent amount of purified recombinant V antigen plus Alhydrogel adjuvant. Similarly, immunization with AdLacZ-pIX/F1 in a prime-boost regimen resulted in significantly enhanced protection of immunized animals compared with immunization with a molar-equivalent amount of purified recombinant F1 protein plus adjuvant. These observations demonstrate that Ad vaccine vectors containing pathogen-specific antigens fused to the pIX capsid protein have strong adjuvant properties and stimulate more robust protective immune responses than equivalent recombinant protein-based subunit vaccines administered with conventional adjuvant, suggesting that F1-and/or V-modified capsid Ad-based recombinant vaccines should be considered for development as anti-plague vaccines.

Gene Therapy to Stimulate Angiogenesis to Treat Diffuse Coronary Artery Disease

Cardiac gene therapy offers a strategy to treat diffuse coronary artery disease (CAD), a disorder with no therapeutic options. The use of genes to revascularize the ischemic myocardium has been the focus of two decades of preclinical research with a variety of angiogenic mediators, including vascular endothelial growth factor, fibroblast growth factor, hepatocyte growth factor, and others encoded by DNA plasmids or adenovirus vectors. The multifaceted challenge for developing efficient induction of collateral vessels in the ischemic heart requires a choice for route of delivery, dosing level, a relevant animal model, duration of treatment, and assessment of phenotype for efficacy. Overall, studies of gene therapy for ischemia in experimental models are very encouraging, with clear evidence of safety and efficacy, strongly supporting the concept that gene therapy to induce angiogenesis is a viable therapeutic approach for CAD. Clinical studies of cardiac gene therapy with angiogenic factors have added substantially to the evidence for efficacy, but definitive studies have not yet led to commercial approval. This review provides the general concepts for angiogenesis-based therapeutic approaches for diffuse CAD and summarizes the results from key studies in the field with recommendations for refinement to a successful product design and evaluation.

Immunogenicity of Seven New Recombinant Yellow Fever Viruses 17D Expressing Fragments of SIVmac239 Gag, Nef, and Vif in Indian Rhesus Macaques

An effective vaccine remains the best solution to stop the spread of human immunodeficiency virus (HIV). Cellular immune responses have been repeatedly associated with control of viral replication and thus may be an important element of the immune response that must be evoked by an efficacious vaccine. Recombinant viral vectors can induce potent T-cell responses. Although several viral vectors have been developed to deliver HIV genes, only a few have been advanced for clinical trials. The live-attenuated yellow fever vaccine virus 17D (YF17D) has many properties that make it an attractive vector for AIDS vaccine regimens. YF17D is well tolerated in humans and vaccination induces robust T-cell responses that persist for years. Additionally, methods to manipulate the YF17D genome have been established, enabling the generation of recombinant (r)YF17D vectors carrying genes from unrelated pathogens. Here, we report the generation of seven new rYF17D viruses expressing fragments of simian immunodeficiency virus (SIV)mac239 Gag, Nef, and Vif. Studies in Indian rhesus macaques demonstrated that these live-attenuated vectors replicated in vivo, but only elicited low levels of SIV-specific cellular responses. Boosting with recombinant Adenovirus type-5 (rAd5) vectors resulted in robust expansion of SIV-specific CD8+ T-cell responses, particularly those targeting Vif. Priming with rYF17D also increased the frequency of CD4+ cellular responses in rYF17D/rAd5-immunized macaques compared to animals that received rAd5 only. The effect of the rYF17D prime on the breadth of SIV-specific T-cell responses was limited and we also found evidence that some rYF17D vectors were more effective than others at priming SIV-specific T-cell responses. Together, our data suggest that YF17D – a clinically relevant vaccine vector – can be used to prime AIDS virus-specific T-cell responses in heterologous prime boost regimens. However, it will be important to optimize rYF17D-based vaccine regimens to ensure maximum delivery of all immunogens in a multivalent vaccine.

Genetic modification of neurons to express bevacizumab for local anti-angiogenesis treatment of glioblastoma

The median survival of glioblastoma multiforme (GBM) is approximately 1 year. Following surgical removal, systemic therapies are limited by the blood–brain barrier. To circumvent this, we developed a method to modify neurons with the genetic sequence for therapeutic monoclonal antibodies using adeno-associated virus (AAV) gene transfer vectors, directing persistent, local expression in the tumor milieu. The human U87MG GBM cell line or patient-derived early passage GBM cells were administered to the striatum of NOD/SCID immunodeficient mice. AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin), an anti-human vascular endothelial growth factor (VEGF) monoclonal antibody, was delivered to the area of the GBM xenograft. Localized expression of bevacizumab was demonstrated by quantitative PCR, ELISA and western blotting. Immunohistochemistry showed that bevacizumab was expressed in neurons. Concurrent administration of AAVrh.10BevMab with the U87MG tumor reduced tumor blood vessel density and tumor volume, and increased survival. Administration of AAVrh.10BevMab 1 week after U87MG xenograft reduced growth and increased survival. Studies with patient-derived early passage GBM primary cells showed a reduction in primary tumor burden with an increased survival. These data support the strategy of AAV-mediated central nervous system gene therapy to treat GBM, overcoming the blood–brain barrier through local, persistent delivery of an anti-angiogenesis monoclonal antibody.

Anti-hIgE gene therapy of peanut-induced anaphylaxis in a humanized murine model of peanut allergy

BACKGROUND Peanuts are the most common food to provoke fatal or near-fatal anaphylactic reactions. Treatment with an anti-hIgE mAb is efficacious but requires frequent parenteral administration. OBJECTIVE Based on the knowledge that peanut allergy is mediated by peanut-specific IgE, we hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector encoding for anti-hIgE would protect against repeated peanut exposure in the host with peanut allergy. METHODS We developed a novel humanized murine model of peanut allergy that recapitulates the human anaphylactic response to peanuts in NOD-scid IL2Rgammanull mice transferred with blood mononuclear cells from donors with peanut allergy and then sensitized with peanut extract. As therapy, we constructed an adeno-associated rh.10 serotype vector coding for a full-length, high-affinity, anti-hIgE antibody derived from the Fab fragment of the anti-hIgE mAb omalizumab (AAVrh.10anti-hIgE). In the reconstituted mice peanut-specific IgE was induced by peanut sensitization and hypersensitivity, and reactions were provoked by feeding peanuts to mice with symptoms similar to those of human subjects with peanut allergy. RESULTS A single administration of AAVrh.10anti-hIgE vector expressed persistent levels of anti-hIgE. The anti-hIgE vector, administered either before sensitization or after peanut sensitization and manifestation of the peanut-induced phenotype, blocked IgE-mediated alterations in peanut-induced histamine release, anaphylaxis scores, locomotor activity, and free IgE levels and protected animals from death caused by anaphylaxis. CONCLUSION If this degree of persistent efficacy translates to human subjects, AAVrh.10anti-hIgE could be an effective 1-time preventative therapy for peanut allergy and possibly other severe, IgE-mediated allergies.

Vaccination with gag, vif, and nef Gene Fragments Affords Partial Control of Viral Replication after Mucosal Challenge with SIVmac239

ABSTRACT Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with “minigenes” encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication. We delivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01 + vaccinees mounted CD8+ T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication. IMPORTANCE Immunodominance poses a major obstacle to the generation of broadly targeted, HIV-specific cellular responses by vaccination. Here we attempted to circumvent this phenomenon and thereby broaden the repertoire of SIV-specific cellular responses by vaccinating rhesus macaques with minigenes encoding fragments of Gag, Vif, and Nef. In contrast to previous mouse studies, this strategy appeared to minimally affect monkey CD8+ T-cell immundominance hierarchies, as seen by the detection of only one subdominant epitope in Mamu-A*01 + vaccinees. This finding underscores the difficulty of inducing subdominant CD8+ T cells by vaccination and demonstrates that strategies other than gene fragmentation may be required to significantly alter immunodominance in primates. Although some of the regimens tested here were extremely immunogenic, vaccine efficacy was limited to a modest reduction in set point viremia after challenge with SIVmac239. No correlates of protection were identified. These results reinforce the notion that vaccine immunogenicity does not predict control of AIDS virus replication.

In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration

Objective: The reprogramming of cardiac fibroblasts into induced cardiomyocyte‐like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors. Methods: Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague‐Dawley rats then underwent coronary ligation and Ad‐mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology. Results: Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)‐treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull‐treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: −0.4% ± 2%; P < .05). Conclusions: Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte‐like cells and improving cardiac function in postinfarct rat hearts. Short‐term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans.