Rapid Cloning of Novel Rhesus Adenoviral Vaccine Vectors

Abstract

Human and chimpanzee adenovirus vectors are being developed to circumvent preexisting antibodies against common adenovirus vectors such as Ad5. However, baseline immunity to these vectors still exists in human populations. Traditional cloning of new adenovirus vaccine vectors is a long and cumbersome process that takes 2 months or more and that requires rare unique restriction enzyme sites. Here we describe a novel, restriction enzyme-independent method for rapid cloning of new adenovirus vaccine vectors that reduces the total cloning procedure to 1 week. We developed 14 novel adenovirus vectors from rhesus monkeys that can be grown to high titers and that are immunogenic in mice. All vectors grouped with the unusual adenovirus species G and show extremely low seroprevalence in humans. Rapid cloning of novel adenovirus vectors is a promising approach for the development of new vector platforms. Rhesus adenovirus vectors may prove useful for clinical development.IMPORTANCE To overcome baseline immunity to human and chimpanzee adenovirus vectors, we developed 14 novel adenovirus vectors from rhesus monkeys. These vectors are immunogenic in mice and show extremely low seroprevalence in humans. Rhesus adenovirus vectors may prove useful for clinical development.

Keywords: adenoviruses; live vector vaccines; rhesus monkey; vaccines.

FIG 1

Phylogenetic analysis. Maximum likelihood phylogenetic trees for rhesus, human, and chimpanzee adenovirus for complete genomes (A) and hexon genes (B) were generated using PhyML 3.1/3.0 aLRT. DNA sequences were aligned and placed into a tree with TreeDyn 198.3. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. (C) Schematic representation of the placement of fiber genes in relation to the locations of the E1, E3, and E4 regions, not drawn to scale.

FIG 2

Adenovirus vector construction. (A) Schematic representation of adenovirus whole-genome fragments generated by PCR for assembly into the AdApter plasmid and cosmid. Matching overhangs of adjacent PCR fragments are indicated by matched patterns. (B) Representative gel pictures of the PCR fragments that are used to assemble the final constructs. (C and D) Screening of AdApter plasmid (C) and cosmid (D) by restriction enzyme digestion with HincII and BsrGI, respectively, reveals a higher percentage of positive clones for the AdApter plasmid than for the cosmid. Positive clones with expected banding patterns are boxed.

FIG 3

Seroprevalence. Seroprevalence to the RhAd vectors was determined in 200 human serum samples from South Africa and Rwanda (A) and 107 SIV-naive rhesus monkeys (B). Titers are graphed as the dilution at which 90% of virus gets neutralized by antibodies present in the serum. The assay sensitivity cutoff is a dilution of 36.

FIG 4

Immunogenicity in mice. (A and B) Mouse T-cell responses are shown by D^b/AL11 CD8⁺ T-cell tetramer binding assays in PBMCs at weekly intervals after immunization with 10⁸ vp or 10⁹ vp (A) and SIVgag-specific effector and memory CD8⁺ T-cell differentiation by KLRG1 and CD127 staining (B). Undifferentiated precursor (KLRG1⁻/CD127⁻), memory precursor (KLRG1⁻/CD127⁺), long-term effector (KLRG1⁺/CD127⁺), and terminal effector (KLRG1⁺/CD127⁻) populations are shown on day 14 and day 28 postimmunization with 10⁹ vp. (C and D) Programmed cell death 1 (PD-1) expression on SIVgag-specific CD8⁺ T cells are shown on day 14 and day 28 postimmunization with 10⁹ vp (C) and ELISPOT responses in splenocytes 4 weeks postimmunization to the complete SIVgag peptide pool, the dominant CD8⁺ T-cell AL11 epitope, and subdominant CD8⁺ T-cell KV9 and CD4⁺ T-cell DD13 epitopes (D). Results are from C56BL/6 immunized mice (n = 4) and a minimum of 2 repeat experiments.

FIG 5

Tissue tropism and receptor use. (A and B) Tropism of adenovirus vectors in rhesus kidney cells (MK2), human retinal cells (ARPE-19), human duodenum adenocarcinoma cells (HuTu80), human lung carcinoma cells (A549), human primary prostate cells (prostate), and human primary bladder cells (bladder) at MOIs of 100 (A) and 1,000 (B). Results were obtained on an LSRII flow cytometer 24 h postinfection and plotted as the percentages of eGFP-positive cells. (C) Receptor assessment in parental HAP1 cells (black), CD46 knockout cells (red), CAR knockout cells (blue), CD55 knockout cells (green), and sialic acid (CMAS) knockout cells (purple). Cells were incubated for 24 h and analyzed by flow cytometry after an infection of 1 h. Percentages of eGFP-positive cells were normalized to 100% infection in parental cells. Reduced infection in the knockout cell lines suggests the lack of an available cellular entry receptor for the corresponding adenovirus.