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. 2019 Aug 27;9(1):12416.
doi: 10.1038/s41598-019-48880-0.

Antibody response against koala retrovirus (KoRV) in koalas harboring KoRV-A in the presence or absence of KoRV-B

O Olagoke  1 , B L Quigley  1 , M V Eiden  2 , P Timms  3
Affiliations

Antibody response against koala retrovirus (KoRV) in koalas harboring KoRV-A in the presence or absence of KoRV-B

O Olagoke et al. Sci Rep. .

Abstract

Koala retrovirus (KoRV) is in the process of endogenization into the koala (Phascolarctos cinereus) genome and is currently spreading through the Australian koala population. Understanding how the koala's immune system responds to KoRV infection is critical for developing an efficacious vaccine to protect koalas. To this end, we analyzed the antibody response of 235 wild koalas, sampled longitudinally over a four-year period, that harbored KoRV-A, and with or without KoRV-B. We found that the majority of the sampled koalas were able to make anti-KoRV antibodies, and that there was a linear increase in anti-KoRV IgG levels in koalas up to approximately seven years of age and then a gradual decrease thereafter. Koalas infected with both KoRV-A and KoRV-B were found to have slightly higher anti-KoRV IgG titers than koalas with KoRV-A alone and there was an inverse relationship between anti-KoRV IgG levels and circulating KoRV viral load. Finally, we identified distinct epitopes on the KoRV envelope protein that were recognized by antibodies. Together, these findings provide insight into the koala's immune response to KoRV and may be useful in the development of a therapeutic KoRV vaccine.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematics showing KoRV genome and relevant segments used in this study (modified from Olagoke et al.). (a) Representative KoRV genome showing coding regions. (b) Segment of the env protein used to produce recombinant protein (rEnv) and synthetic peptide (MPER peptide), and for epitope mapping. The numbers shown represent corresponding amino acid number (GenBank: AF151794). (c) Layout of the transmembrane subunit of KoRV envelope protein showing important regions. The numbers represent 15mer amino acids peptides with three amino acids offset. FP = fusion peptide, FPPR = fusion peptide proximal region, HR1 = heptad repeat 1, IS = immunosuppressive domain, HR2 = heptad repeat 2, MPER = membrane proximal external region, TM = transmembrane region.
Figure 2
Figure 2
Anti-KoRV rEnv IgG levels expressed as end-point titers (EPT) in serum samples of KoRV-A positive koalas (n = 235). The green line indicates the assay background.
Figure 3
Figure 3
Association between anti-KoRV IgG levels expressed as end-point titers (EPT) in koala serum and age of the koala. (A) Association was tested using regression analysis (second order polynomial) at a population level in 207 koalas. The level of significance was measured as p < 0.001. (B,C) are examples of serum IgG measured in young and old koalas respectively over a minimum of three years. All 14 koalas are shown in Supplementary Fig. 2.
Figure 4
Figure 4
Anti-KoRV rEnv IgG levels expressed as end-point titers (EPT) in serum samples from KoRV-B negative (n = 186) and KoRV-B positive (n = 42) koalas compared and presented as mean ± SD. The level of significance was measured using student’s T-test (p < 0.05).
Figure 5
Figure 5
Relationship between plasma KoRV viral RNA load (copies/mL) as measured by qPCR in plasma of koalas and serum anti-KoRV IgG levels (EPT) in paired plasma and serum samples. KoRV-B negative koalas (n = 27) are shown in black circles while KoRV-B positive koalas (n = 9) are shown in red circles (R2 = 0.53, p = 0.011, n = 34).
Figure 6
Figure 6
p15E ectodomain B cell epitope mapping in two groups of KoRV-A positive koalas sampled over two time points: (A) dependent juveniles (n = 3) (0.9–1 year, white bars) and again as adults (4.5 years, black bars), and (B) older koalas (n = 3) (9–10 years, white bars) and again at 12–12.9 years (black bars). The numbers on the x-axis represent 15-mer amino acids peptides with three amino acids offsets spanning the ectodomain of KoRV-A env protein and the boxes represent epitopes of interest. (C) Comparison of amino acid sequences around distinct epitopes (colored boxes and lines) on KoRV p15E ectodomain among different KoRV subtypes. Conserved amino acid residues are indicated by *. FP = fusion peptide, FPPR = fusion peptide proximal region, HR1 = heptad repeat 1, IS = immunosuppressive domain, HR2 = heptad repeat 2, MPER = membrane proximal external region, TM = transmembrane region.
Figure 7
Figure 7
Comparison of p15E ectodomain B cell epitopes recognized in (A) non-vaccinated northern koalas harbouring KoRV-A (n = 12) and (B) vaccinated southern koalas believed to be infected with exogenous KoRV-A (n = 3). The numbers on the x-axis represent 15-mer amino acids peptides with three amino acids offsets spanning the ectodomain of KoRV-A env protein and the colored boxes represent regions of interest. FP = fusion peptide, FPPR = fusion peptide proximal region, HR1 = heptad repeat 1, IS = immunosuppressive domain, HR2 = heptad repeat 2, MPER = membrane proximal external region, TM = transmembrane region.
Figure 8
Figure 8
(A) MPER IgG levels measured at 450 nm OD in serum samples of koalas (n = 197) harboring KoRV-A. The green line indicates the assay background (OD of 0.12, determined from taking the mean plus two times the standard deviation of three KoRV negative koalas). (B) Correlation between the levels of MPER IgG (measured at 450 nm OD) and rEnv IgG (measured at 450 nm OD) in koalas (R2 = 0.44, p = 0.0001, n = 197).
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