Chimeric Ad5.F35 vector evades anti-adenovirus serotype 5 neutralization opposing GUCY2C-targeted antitumor immunity

BACKGROUND: Adenovirus serotype 5 (Ad5) is a commonly used viral vector for transient delivery of transgenes, primarily for vaccination against pathogen and tumor antigens. However, endemic infections with Ad5 produce virus-specific neutralizing antibodies (NAbs) that limit transgene delivery and constrain target-directed immunity following exposure to Ad5-based vaccines. Indeed, clinical trials have revealed the limitations that virus-specific NAbs impose on the efficacy of Ad5-based vaccines. In that context, the emerging focus on immunological approaches targeting cancer self-antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectors. METHODS: Here, we evaluated the ability of a chimeric adenoviral vector (Ad5.F35) derived from the capsid of Ad5 and fiber of the rare adenovirus serotype 35 (Ad35) to induce immune responses to the tumor-associated antigen guanylyl cyclase C (GUCY2C). RESULTS: In the absence of pre-existing immunity to Ad5, GUCY2C-specific T-cell responses and antitumor efficacy induced by Ad5.F35 were comparable to Ad5 in a mouse model of metastatic colorectal cancer. Furthermore, like Ad5, Ad5.F35 vector expressing GUCY2C was safe and produced no toxicity in tissues with, or without, GUCY2C expression. Importantly, this chimeric vector resisted neutralization in Ad5-immunized mice and by sera collected from patients with colorectal cancer naturally exposed to Ad5. CONCLUSIONS: These data suggest that Ad5.F35-based vaccines targeting GUCY2C, or other tumor or pathogen antigens, may produce clinically relevant immune responses in more (≥90%) patients compared with Ad5-based vaccines (~50%)

Can. Imp. Bank of Comm. : Yonge & Pleasant Blvd Branch : Robbery

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An improved method to quantitate mature plant microRNA in biological matrices using modified periodate treatment and inclusion of internal controls

<div><p>MicroRNAs (miRNAs) ubiquitously exist in microorganisms, plants, and animals, and appear to modulate a wide range of critical biological processes. However, no definitive conclusion has been reached regarding the uptake of exogenous dietary small RNAs into mammalian circulation and organs and cross-kingdom regulation. One of the critical issues is our ability to assess and distinguish the origin of miRNAs. Although periodate oxidation has been used to differentiate mammalian and plant miRNAs, validation of treatment efficiency and the inclusion of proper controls for this method were lacking in previous studies. This study aimed to address: 1) the efficiency of periodate treatment in a plant or mammalian RNA matrix, and 2) the necessity of inclusion of internal controls. We designed and tested spike-in synthetic miRNAs in various plant and mammalian matrices and showed that they can be used as a control for the completion of periodate oxidation. We found that overloading the reaction system with high concentration of RNA resulted in incomplete oxidation of unmethylated miRNA. The abundant miRNAs from soy and corn were analyzed in the plasma, liver, and fecal samples of C57BL/6 mice fed a corn and soy-based chow diet using our improved methodology. The improvement resulted in the elimination of the false positive detection in the liver, and we did not detect plant miRNAs in the mouse plasma or liver samples. In summary, an improved methodology was developed for plant miRNA detection that appears to work well in different sample matrices.</p></div

Utilization of nitrogen sources by H99 and R265.

<p>5 µl of cell suspensions at OD₆₀₀nm of 10, 0.1 and 0.001 were spotted on 2% glucose YNB agar with 10 mM of the indicated nitrogen source and incubated for 5–7 days at 30°C. Amino acids differentially utilized by the two species are italicized and underlined.</p

Disruption of <i>GAT1</i> reduced ability to utilize all nitrogen sources.

<p>Wild type and <i>gat1Δ</i> strains were grown for 5–7 days at 30°C on 2% glucose YNB with 10 mM of different nitrogen sources. 5 µl cell suspensions at OD600nm of 10, 0.1 and 0.001 were spotted on the media. YPD and Blank (no nitrogen source) served as positive and negative control respectively.</p

Disruption of <i>GAT1</i> enhanced melanin synthesis and <i>LAC1</i> expression in both species but capsule size increased only in R265.

<p>A) Cells were grown in melanin induction media using 10 mM proline as the nitrogen source. Melanin production was assayed by measuring the absorbance at 475 nm and B) <i>LAC1</i> expressions were quantified by real time PCR. C) Micrograph of cell grown in capsule induction media. D) The relative capsule size was measured in >100 cells for each strain grown in capsule induction media. Experiments were carried out in triplicates. * <i>p</i>≤0.05 by the student t-test. Error bars = 2 standard deviation.</p

Utilization of different nitrogen sources according to molecular type.

<p><i>C. n.</i> = <i>Cryptococcus neoformans</i> and <i>C.g.</i> = <i>Cryptococcus gattii</i>. Bold italic are test giving 100% correlation with either species.</p

Virulence of <i>H99gat1Δ</i> was minimally enhanced while of <i>R265gat1Δ</i> was significantly reduced in murine inhalation model.

<p>50,000 cells from each strain were inoculated intrapharyngeally to 10 Balb/c mice per group. Mice were fed with food and water ad libitum.</p

List of strains and their ability to utilize different nitrogen sources.

<p>List of strains and their ability to utilize different nitrogen sources.</p

The role of <i>GAT1</i> in cell growth.

<p>A) <i>GAT1</i> is not associated with the growth rate of both species. Cell suspensions of 2 OD₆₀₀ were spotted on YPD agar in three 10 fold dilutions and incubated for three days at the designated temperatures B) Similar spot assays were carried out on YPD with or without caffeine and plates were incubated at 30°C for 3 days. Experiments were carried out in duplicates.</p