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. 2015 Feb;89(3):1512-22.
doi: 10.1128/JVI.02950-14. Epub 2014 Nov 19.

Construction and evaluation of novel rhesus monkey adenovirus vaccine vectors

Peter Abbink  1 , Lori F Maxfield  1 , David Ng'ang'a  1 , Erica N Borducchi  1 , M Justin Iampietro  1 , Christine A Bricault  1 , Jeffrey E Teigler  1 , Stephen Blackmore  1 , Lily Parenteau  1 , Kshitij Wagh  2 , Scott A Handley  3 , Guoyan Zhao  3 , Herbert W Virgin  3 , Bette Korber  2 , Dan H Barouch  4
Affiliations

Construction and evaluation of novel rhesus monkey adenovirus vaccine vectors

Peter Abbink et al. J Virol. 2015 Feb.

Abstract

Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. The phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. Here we describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved to have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.

Importance: Although there have been substantial efforts in the development of vaccine vectors from human and chimpanzee adenoviruses, far less is known about rhesus monkey adenoviruses. In this report, we describe the isolation and vectorization of three novel rhesus monkey adenoviruses. These vectors exhibit virologic and immunologic characteristics that make them attractive as potential candidate vaccine vectors for both HIV-1 and other pathogens.

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Figures

FIG 1
FIG 1
Adenovirus identification and genomic structure. (A) PCR-amplified regions in the first crude lysate after infection of cells with filtered stool samples (crude lysate), 4 separate plaques after the second round of plaque purification, and CsCl density gradient-purified virus. Listed are the targeted regions in the adenovirus genome and their expected band sizes. First lane, a 1-kb-plus DNA ladder. (B) The layout of the early (E) and late (L) genes of the novel simian adenoviruses is similar to that of known human adenoviruses. (C) The AdApter plasmid comprises the left end of the adenovirus genome in which E1 is replaced with a transgene cassette (TGC). The cosmid contains the remaining right end of the adenovirus genome in which E3 is deleted. Ψ, virus packaging signal.
FIG 2
FIG 2
Maximum likelihood phylogenetic tree of adenovirus genomes. Full-length genomes from classified (species A to G) and unclassified human and simian adenoviruses, in addition to the novel vectors, were used to infer an ML phylogenetic tree. The different species are highlighted by colored rectangles (sA, simian species A), and in the case of simian vectors, the host species are noted (Rh, rhesus macaque; Cy, cynomolgus macaque; Go, gorilla; Ve, vervet monkey; Ba, baboon; Bo, bonobo; Ch, chimpanzee). The novel vectors were closest to species G adenoviruses (red arrows). Other vaccine candidates are highlighted with a V.
FIG 3
FIG 3
Novel vectors are most similar to species G throughout their genomes. (A) Gene map for the novel vector RhAd52. For simplicity, some genes are not shown. (B to D) The similarity of the sequences of the novel vectors to the consensus from each of species A to G is shown in sliding windows of 1 kb along the genome. The similarity score is shown as a fraction and is plotted at the midpoint of each sliding window. Each subplot has 2 horizontal lines of dots on the top. The lower line shows the color of the best matching consensus sequence for a sliding window. The consensus species G sequence is shown in red. The top line shows a dot only when the best match is significant, at a P value of <0.01. Con, consensus.
FIG 4
FIG 4
Seroprevalence of antibodies to the novel rhesus monkey adenoviruses. The seroprevalence of the novel rhesus monkey adenoviruses compared to that of human Ad5 and Ad26 and ChAd24 in 80 South African and 64 Rwandan human serum samples (A) and in 108 naive rhesus monkey serum samples (B) was determined. Undetectable and low titers are shown in yellow and red, respectively; medium and high titers are shown in blue and black, respectively.
FIG 5
FIG 5
Transgene expression by novel rhesus monkey adenovirus vectors. A Western blot of lysates of cells collected 48 h postinfection with HIV-1 Env-expressing adenoviruses is shown. MAb 5F3 was used as the primary antibody, and purified HIV-1 Env protein was used as the positive control.
FIG 6
FIG 6
Humoral immune responses in mice. BALB/c mice (n = 4) received a single immunization with 108 or 109 vp of Ad vectors expressing HIV-1 459C Env gp140 at day 0. Immunization with purified protein was used as the positive control. The titers of antibodies to heterologous HIV-1 C97ZA012 and Mosaic Env gp140 as well as to homologous 459C Env gp140 in serum were determined by ELISA on day 0 and day 28. Red lines, medians; dotted lines, titer cutoff 2 times the average background titer.
FIG 7
FIG 7
Cellular immune responses in mice. (A) A single immunization of C57BL/6 mice with 1 × 109, 1 × 108, or 1 × 107 vp of vectors expressing SIV Gag at day 0. Gag-specific CD8+ T cells were detected in serum by Db/AL11 tetramer binding assays at weekly intervals. (B) Spleens were harvested at day 28 and evaluated by IFN-γ ELISPOT assays after stimulation with the DDR13, KV9, and AL11 epitopes as well as a whole SIV Gag peptide pool. SFC, number of spot-forming cells.
FIG 8
FIG 8
Receptor binding assays. A549 cells were infected with eGFP-labeled adenovirus vectors alone or after 1 h preincubation with anti-CAR (E1-1, 3C100) or anti-CD46 (M177, MEM-258) antibodies. Cells were washed, harvested, fixed after a 48-h incubation period, and analyzed by flow cytometry. Antibody concentrations are in μg/ml. The value for transduced cells in the absence of antibody was normalized to 100%.
FIG 9
FIG 9
Cytokine and chemokine responses elicited in rhesus monkey PBMCs. PBMCs (n = 3) were stimulated with 1 × 103 vp/cell of various adenovirus vectors and incubated for 24 h. Cytokine and chemokine responses were measured by Luminex assays. Lipopolysaccharide at 1 ng/ml was used as a positive control. Statistical analysis was done with one-way analysis of variance between all groups. ***, P ≤ 0.0001; *, P < 0.03.
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