Barry, Michael A.

Most infections occur at mucosal surfaces. Providing a barrier of protection at these surfaces may be a useful strategy to combat the earliest events in infection when there are relatively few pathogens to address. The majority of vaccines are delivered systemically by the intramuscular (IM) route. While IM vaccination can drive mucosal immune responses, mucosal immunization at intranasal (IN) or oral sites can lead to better immune responses at mucosal sites of viral entry. In macaques, IN immunization with replicating single-cycle adenovirus (SC-Ads) and protein boosts generated favorable mucosal immune responses. However, there was an apparent “distance effect” in generating mucosal immune responses. IN immunization generated antibodies against HIV envelope (env) nearby in the saliva, but weaker responses in samples collected from the distant vaginal samples. To improve on this, we tested here if SC-Ads expressing genetic adjuvants could be used to amplify antibody responses in distant vaginal samples when they are codelivered with SC-Ads expressing clade C HIV env immunogen. SC-Ads env 1157 was coadministered with SC-Ads expressing 4-1BBL, granulocyte macrophage colony-stimulating factor (GMCSF), IL-21, or Clostridoides difficile (C. diff.) toxin fragments by IN or IM routes. These data show that vaginal antibody responses were markedly amplified after a single immunization by the IN or IM routes, with SC-Ad expressing HIV env if this vaccine is complemented with SC-Ads expressing genetic adjuvants. Furthermore, the site and combination of adjuvants appear to “tune” these antibody responses towards an IgA or IgG isotype bias. Boosting these priming SC-Ad responses with another SC-Ad or with SOSIP native-like env proteins markedly amplifies env antibody levels in vaginal washes. Together, this data may be useful in informing the choice of route of delivery adenovirus and peptide vaccines against HIV-1.

In the murine venous thrombosis model induced by ligation of the inferior vena cava (IVCL), genetic deficiency of heme oxygenase-1 (HO-1) increases clot size. This study examined whether induction of HO-1 or administration of its products reduces thrombosis. Venous HO-1 upregulation by gene delivery reduced clot size, as did products of HO activity, biliverdin, and carbon monoxide. Induction of HO-1 by hemin reduced clot formation, clot size, and upregulation of plasminogen activator inhibitor-1 (PAI-1) that occurs in the IVCL model, while leaving urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) expression unaltered. The reductive effect of hemin on clot size required HO activity. The IVCL model exhibited relatively high concentrations of heme that peaked just before maximum clot size, then declined as clot size decreased. Administration of hemin decreased heme concentration in the IVCL model. HO-2 mRNA was induced twofold in the IVCL model (vs. 40-fold HO-1 induction), but clot size was not increased in HO-2 −/− mice compared with HO-2 +/+ mice. Hemopexin, the major heme-binding protein, was induced in the IVCL model, and clot size was increased in hemopexin −/− mice compared with hemopexin +/+ mice. We conclude that in the IVCL model, the heme-degrading protein HO-1 and HO products inhibit thrombus formation, as does the heme-binding protein, hemopexin. The reductive effects of hemin administration require HO activity and are mediated, in part, by reducing PAI-1 upregulation in the IVCL model. We speculate that HO-1, HO, and hemopexin reduce clot size by restraining the increase in clot concentration of heme (now recognized as a procoagulant) that otherwise occurs. NEW & NOTEWORTHY This study provides conclusive evidence that two proteins, one heme-degrading and the other heme-binding, inhibit clot formation. This may serve as a new therapeutic strategy in preventing and treating venous thromboembolic disease.

Adenoviruses (Ads) are robust vectors for therapeutic applications and vaccines, but their use can be limited by differences in their in vitro and in vivo pharmacologies. This review emphasizes that there is not just one Ad, but a whole virome of diverse viruses that can be used as therapeutics. It discusses that true vector targeting involves not only retargeting viruses, but importantly also detargeting the viruses from off‐target cells.

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale . Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

There are many kidney diseases that might be addressed by gene therapy. However, gene delivery to kidney cells is inefficient. This is due, in part, to the fact that the kidney excludes molecules above 50 kDa and that most gene delivery vectors are megaDaltons in mass. We compared the ability of adeno-associated virus (AAV), adenovirus (Ad), and lentiviral (LV) vectors to deliver genes to renal cells. When vectors were delivered by the intravenous (IV) route in mice, weak luciferase activity was observed in the kidney with substantially more in the liver. When gene delivery was observed in the kidney, expression was primarily in the glomerulus. To avoid these limitations, vectors were injected directly into the kidney by retrograde ureteral (RU) and subcapsular (SC) injections in mice. Small AAV vectors transduced the kidney, but also leaked from the organ and mediated higher levels of transduction in off-target tissues. Comparison of AAV2, 6.2, 8, and rh10 vectors by direct kidney injection demonstrated highest delivery by AAV6.2 and 8. Larger Ad and LV vectors transduced kidney cells and mediated less off-target tissue transduction. These data demonstrate the utility of direct kidney injections to circumvent the kidney size exclusion barrier. They also identify the effects of vector size on on-target and off-target transduction. This lays the foundation for the use of different vector platforms for gene therapy of diverse kidney diseases.

Significance Provision of sufficient activating signals can drive self-tolerant T cells to cross-react on self-peptide-MHC in a breakdown of tolerance. This approach has been applied in the development of heteroclitic peptide vaccines, which retain sufficient similarity to self to allow reactivation of effector cells on unaltered self-epitopes but require detailed knowledge about the behavior of individual altered self-peptide antigens. We designed altered-self MHC mutant antigen-presenting molecules to drive CTL capable of cross-reacting on self-peptide-MHC and demonstrate the breaking of self-tolerance to defined and undefined self-antigens. These findings bear particular importance in the field of cancer immunotherapy in which breaking tolerance to weakly antigenic cancer-associated antigens is fundamental to vaccination strategies.

HIV-1 infections usually start at a mucosal surface after sexual contact. Creating a barrier of protection at these mucosal sites may be a good strategy for to protect against HIV-1 infections. While HIV-1 enters at mucosa, most vaccines are not delivered here. Most are instead injected into the muscle, a site well distant and functionally different than mucosal tissues. This study tested if delivering HIV vaccines at mucosa or in the muscle makes a difference in the quality, quantity, and location of immune responses against the virus. These data suggest that there are indeed advantages to educating the immune system at mucosal sites with an HIV-1 vaccine.

Abstract Oncolytic viruses hold promise as “self-amplifying” cancer therapies wherein a virally killed cell can produce thousands of new viral “drugs” that can kill more cancer cells. Oncolytic viruses may also serve to reawaken or initiate immune responses against cancers by virtue of their abilities to lyse cells, liberate cancer antigens, and provide viral DNA and proteins as immune adjuvants. Oncolytics can also be armed with cytokine or checkpoint inhibitors to amplify antitumor immune responses. Adenoviruses (Ads) are one family of oncolytic viruses. Most human studies have used human Ad serotype 5 (Ad5). Unfortunately, most patients are already immune to Ad5, increasing the likelihood that the agent will be neutralized if used as a cancer therapy. In this work, low seroprevalence Ad6 and Ad57 oncolytic viruses were tested as intravenous therapies against distant prostate cancer sites. After prostate cell infection in vitro, Ad viruses executed these cells efficiently and generated 10,000 to 100,000 progeny infectious viruses. This genome replication also amplified transgene protein production by 100 to 1,000-fold when compared to nonreplicating Ad vectors. Ad5, Ad6, and Ad57 were injected intravenously a single time in nude mice bearing human prostate tumors, and toxicity and efficacy were assessed. Ad6 and 57 produced 30-fold lower liver damage and less toxicity than Ad5. Ad6 and Ad57 significantly repressed the growth of androgen-resistant human DU145 prostate tumors and androgen-sensitive LNCaP prostate tumors and extended survival after single intravenous injection in mice. Ad6 and Ad57 E1 protein were also modified to protect normal cells from viral oncolysis while allowing the virus to amplify and kill prostate cancer cells having perturbations in p300 and pRB pathways. These and other data suggest that targeted and untargeted low seroprevalent Ad6 and Ad57 have better efficacy and safety than the benchmark oncolytic virus Ad5 for systemic therapy of advanced prostate cancer. Citation Format: Tien Nguyen, Michael A. Barry. Systemic oncolytic virotherapy for prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A062.