Woolly Monkey-HBV Infection in Squirrel Monkeys as a Surrogate Nonhuman Primate Model of HBV Infection

Abstract

Development of curative therapies for chronic hepatitis B virus (HBV) infection will likely require new animal models. Here, we evaluate HBV infection in squirrel monkeys based on the high-sequence homology of the HBV receptor, Na+/taurocholate co-transporting peptide (NTCP), between humans and squirrel monkeys. HBV PreS1 peptide was examined for binding human and squirrel monkey NTCP. Immunodeficient Fah ^-/- , NOD, Rag1 ^-/- , Il2Rg ^null (FNRG) mice engrafted with human or squirrel monkey hepatocytes were challenged with HBV or Woolly Monkey HBV (WMHBV). In addition, adult squirrel monkeys were inoculated with HBV, WMHBV, adeno-associated virus containing an infectious genome of HBV (AAV-HBV), and AAV-WMHBV. Finally, neonate squirrel monkeys were assessed for the potential of chronic infection with WMHBV. PreS1 peptide efficiently bound to human and squirrel monkey NTCP but not to mouse or capuchin NTCP. FNRG mice engrafted with squirrel monkey hepatocytes were susceptible to infection by WMHBV but not human HBV. Similarly, adult squirrel monkeys could be infected with WMHBV but not human HBV, whereas chimeric mice engrafted with human hepatocytes were susceptible to HBV but not WMHBV. Infection of squirrel monkeys with AAV-WMHBV yielded maximum viremia of 10⁸ genomes/mL with detectable virus for up to 8 months. Notably, covalently closed circular DNA was detected in the liver of these animals. Infection of neonates with WMHBV led to detectable viremia for up to 6 months. Conclusions: Adult and neonate squirrel monkeys exhibited prolonged WMHBV viremia lasting 6-8 months. This is greater than twice the duration of viremia achieved in other nonhuman primates and suggests that squirrel monkeys may be a suitable model for testing HBV therapeutics.

Figure 1

(A) Amino acid sequence alignment of NTCP from putative HBV animal models. The gray bar highlights the amino acid at position 158, which is critical for mediating binding to HBV. (B) Phylogenetic tree depicting evolutionary distance between species from (1A) based on difference in amino acid sequence of NTCP.

Figure 2

Myrcludex B binds squirrel monkey NTCP. HEK293 cells were transfected with protein expression vectors that express species‐specific NTCPs. (A) Two days after transfection, the cells were either lysed for western blot analysis of NTCP expression. (B) NTCP‐expressing HEK293 cells were incubated with fluorescently labeled Myrcludex B (left column). Fluorescent Myrcludex B binding could be blocked by pre‐incubation with unlabeled Myrcludex B (right column). Abbreviations: DAPI, 4´,6‐diamidino‐2‐phenylindole (a type of stain).

Figure 3

Squirrel monkey liver chimeric FNRG mice support WMHBV infection and transcription. FNRG mice were engrafted with either primary human or squirrel monkey hepatocytes and were subsequently challenged with HBV or WMHBV. Primary hepatocyte engraftment was determined by ELISA for circulating albumin of the respective species. (A) Albumin levels for FNRG mice engrafted with primary human hepatocytes. (B) Albumin levels for FNRG mice engrafted with squirrel monkey hepatocytes. The red arrow depicts when mice were challenged with either HBV or WMHBV. The dotted line indicates the limit of detection for the assay. Quantification of total HBV and WMHBV DNA (C) and pgRNA (D) in liver tissues from human and squirrel monkey liver chimeric mice that had been challenged with either HBV or WMHBV. Abbreviation: Alb, albumin.

Figure 4

AAV‐WMHBV elicits early high‐titer viremia that remains detectable for up to 8 months. Serum viral titers and ALT levels from adult squirrel monkeys infected with HBV (A), WMHBV (B), AAV‐HBV (C), or AAV‐WMHBV (D). Accompanying tables show ELISA measurements of HBsAg (ng/mL) and HBeAg (ng/mL) as well as the presence of anticore antibodies. The line indicates the limit of detection for the PCR assays, 10² GE/mL of serum.

Figure 5

HBV cccDNA and viral transcripts are detectable in the liver of AAV‐WMHBV‐infected squirrel monkeys. Squirrel monkeys that had been infected with AAV‐HBV or AAV‐WMHBV had viral DNA and RNA isolated from the liver. (A) HBV and WMHBV genome copies were measured from the total liver DNA of the indicated adult squirrel monkeys at weeks 4 and 14. The line indicates the limit of detection for the PCR assays, 10¹ GE/$\mathrm{\mu }$g of DNA. (B) RNA transcripts from liver tissue were measured by RT‐PCR. The line indicates the limit of detection for the PCR assay, 10¹ GE/$\mathrm{\mu }$g of RNA. (C) DNA from the liver DNA were analyzed by southern blot for viral DNA. Arrow highlighting the 2kB band indicates the predicted band size for cccDNA. (D) DNA from liver biopsies was treated with T5 nuclease to digest non‐supercoiled DNA and then analyzed by southern blot. cccDNA is resistant to T5 digestion. (E) In situ hybridization (RNAscope) for woolly monkey RNA in formalin‐fixed liver sections from squirrel monkey 36244, before and after infection with AAV‐WMHBV.

Figure 6

Changes in IFN gene expression and markers of cell death and proliferation in the liver of AAV‐WMHBV‐infected monkeys. (A) TaqMan RT‐PCR was performed on total liver RNA from four control animals (C) and two animals infected with AAV‐WMHBV (36244, 36245) at 4 and 14 weeks following infection. Primers and probes for squirrel monkey IFN gamma, CXCL9 (i.e., Mig), and CXCL10 (i.e., IP‐10) were designed based on the genomic sequence of squirrel monkeys as described in the Materials and Methods section. The values for infected animals are expressed as a fold change in comparison to the controls. C is the average of four animals and is expressed as a fold change of 1, no fold change, with the range shown. (B) Immunohistochemical staining of liver sections from an AAV‐WMHBV‐infected monkey at week 4 and 14 for the cell death marker–activated caspase‐3 (left, upper, and lower panels) and for the cell proliferation maker Ki67 (right, upper, and lower panels).

Figure 7

Squirrel monkeys infected with WMHBV as neonates remain viremic for up to 6 months. Quantitation of WMHBV genomes in serum from six squirrel monkey neonates infected with WMHBV.