Isolation and Characterization of Adenoviruses Persistently Shed from the Gastrointestinal Tract of Non-Human Primates

Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors

Localization of LacZ expression in injected muscle following IM injection of AAV8 in C57BL/6 mice.

<p>Localization of LacZ expression in injected muscle was determined at day 21 post-vector administration in C57BL/6 mice. Vector was administered at a dose of 10¹⁰ GC as two 25 µl injections into the right and left legs (A, B) or as one 2 µl injection (C, D). A lower dose of 10⁹ GC of vector was administered as two 25 µl injections into the right and left legs (E, F) or as one 2 µl injection (G, H). Three sections were taken throughout the injected gastrocnemius muscle of one representative animal per group (A, C, E and G) with one representative liver section per group (B, D, F and H) (n = 4/group).</p

Liver expression following IM vector administration in mice.

<p>Visualization of differential ffLuc expression patterns using Xenogen whole-body bioluminescent imaging on day 7 post-IM administration of 10¹⁰ GC AAV8.CMV.ffLuc (A) or AAV8.TBG.ffLuc (B) to C57BL/6 mice. Vector was administered as one 10 µl injection into the gastrocnemius muscle of the right leg. (C) ffLuc expression was quantified at day 28 post-vector administration by ffLuc tissue assays. ffLuc expression measured as RLU normalized to the total protein concentration of the organ. Data are presented as fold change over background where background was RLU/total protein of organ (g) in control tissues from un-injected mice. (D) Comparison of expression of 201Ig IA from the CMV and TBG promoters following a 10 µl IM injection in RAG KO mice. Expression of 201Ig IA in serum was measured by ELISA. Values are expressed as mean ± SEM (n = 4/group). ***<i>p</i><0.001.</p

Effect of microRNA target sites on localization and expression level following IM injection of AAV8 in mice.

<p>Differential ffLuc expression patterns were visualized on day 7 post-IM administration of 10¹⁰ GC AAV8.CMV.ffLuc (A), AAV8.CMV.ffLuc.miR-122 (B) or AAV8.CMV.ffLuc.miR-206 (C). ffLuc expressing vectors were administered to C57BL/6 mice in two 10 µl injections into the right and left gastrocnemius muscles. (D) Liver and muscle expression of ffLuc was quantified separately on day 28 post-vector administration. Following substitution of ffLuc for 201Ig IA, expression of 201Ig IA was determined in RAG KO mice. Mice were injected with 10¹¹ GC of vectors, administered as described previously and expression of 201Ig IA in serum was measured by ELISA (E). GC of the 201Ig IA vectors present in the liver of RAG KO mice at day 90 post-vector administration was determined (F). Values are expressed as mean ± SEM (n = 3/group). *<i>p</i><0.05, ***<i>p</i><0.001.</p

Translation of influence of injection volume to a large animal model, the rhesus macaque.

<p>RAG KO mice were injected with 10¹⁰ GC AAV8.CMV.201Ig IA either by IM or IV injection; tissues were harvested on day 56 and analyzed for vector genome copies (GC), quantified as GC/diploid genome in (A) liver and (B) muscle. (C) Biodistribution of AAV8 vector was determined on day 90 post-vector administration in a rhesus macaque. Vector was administered at a dose of 3×10¹² GC/kg by IM injection into the vastus lateralis muscle of both right and left legs as 1 ml injections per kg body weight (vector concentration of 3×10¹² GC/ml). DNA and RNA were extracted for quantification of GC (open bars) and transcript levels of 201Ig IA (closed bars), respectively. Values for muscle are the average of measurements at 12 sites throughout the injected muscle and liver is the average of the four lobes, which were quantified separately. There was no detectable GC or RNA in control (un-injected) muscle samples. LN, lymph node; Ax, axillary; In, inguinal; Mes, mesenteric; Il, iliac; Pop, popliteal. (D) Time course of expression of 201Ig IA in serum. (E) Rhesus macaques were injected IM with 3×10¹¹ GC/kg of AAV8.CMV.201Ig IA, as either 1 ml vector injections per kg body weight (3×10¹¹ GC/ml) or 0.1 ml injection per kg body weight (3×10¹² GC/ml) (n = 2/group). Expression of 201Ig IA was measured in serum by ELISA and values are expressed as mean ± SEM. *<i>p</i><0.05.</p

Comparison of expression from the liver-specific TBG promoter following IM and IV vector administration in RAG KO mice.

<p>Expression on day 28 post-vector administration of 201Ig IA (A), 2.10A mAb (B), VRC01 mAb (C), and PG9 mAb (D) in RAG KO mice. Expression following IM injection of vectors with either the CMV or TBG promoter was compared to expression levels produced following IV injection of the TBG vector. Mice were injected IM with two 15 µl injections into the right and left gastrocnemius muscles. IV injections were performed as a 100 µl injection via the tail vein. Mice were administered with a dose of either 10¹⁰ GC or 10¹¹ GC, numbers indicate dose ×10¹¹ GC. ND, not determined. Expression was measured in serum by ELISA and values are expressed as mean ± SEM (n = 3/group). ***<i>p</i><0.001.</p