Brief Communication

Are there advantages in the use of specificpathogen-free baboons in pig organxenotransplantation models?

Zhou H, Iwase H, Wolf RF, Ekser B, Ezzelarab M, Hara H, White G,Cooper DKC. Are there advantages in the use of specific pathogen-freebaboons in pig organ xenotransplantation models?Xenotransplantation 2014: 21: 287–290. © 2014 John Wiley & Sons A/S.

Abstract: Baboons have natural antibodies against pig antigens. Wehave investigated whether there are differences in anti-non-Gal pig anti-body levels between baboons maintained under specific pathogen-free(SPF) conditions and those housed under conventional conditions (non-SPF) that might be associated with improved outcome after pig-to-baboon organ transplantation. Baboons (n = 40) were housed indoors(SPF n = 8) or in indoor/outdoor pens (non-SPF n = 32) in colonies ofsimilar size and structure. Non-SPF colonies harbor a number of patho-gens common to non-human primate species, whereas many of thesepathogens have been eliminated from the SPF colony. Complete bloodcell counts (CBC), blood chemistry, and anti-non-Gal IgM and IgG lev-els were monitored. There were no significant differences in CBC orblood chemistry between SPF and non-SPF baboons. Anti-non-GalIgM levels were significantly lower in the SPF baboons than in the non-SPF baboons (MFI 7.1 vs. 8.8, P < 0.05). One SPF and two non-SPFbaboons had an MFI >20; if these three baboons are omitted, the meanMFIs were 4.8 (SPF) vs. 7.5 (non-SPF) (P < 0.05). Anti-non-Gal IgGwas minimal in both groups (MFI 1.0 vs. 1.0). As their levels of anti-non-Gal IgM are lower, baboons maintained under SPF conditionsmay be beneficial for xenotransplantation studies as the initial bindingof anti-pig IgM to an a1,3-galactosyltransferase gene-knockout pigorgan may be less, thus resulting in less complement and/or endothelialcell activation. However, even under identical SPF conditions, an occa-sional baboon will express a high level of anti-non-Gal IgM, the reasonfor which remains uncertain.

Huidong Zhou,1,2* Hayato Iwase,1*Roman F. Wolf,3 Burcin Ekser,1

Mohamed Ezzelarab,1 Hidetaka Hara,1

Gary White3 and David K.C. Cooper11Thomas E. Starzl Transplantation Institute,University of Pittsburgh, Pittsburgh, PA, USA,2Center for Kidney Transplantation, SecondAffiliated Hospital of the University of South China,Hengyang, Hunan, China, 3University of OklahomaHealth Sciences Center, Oklahoma City, OK, USA

Key words: antibody – anti-pig – baboon – pig –specific pathogen-free – xenotransplantation –a1,3-galactosyltransferase gene-knockout

Abbreviations: Gal, galactose-a1,3-galactose; GTK,Oa1,3-galactosyltransferase gene-knockout; SPF,specific pathogen-free

Address reprint requests to David K.C. Cooper,Thomas E. Starzl Transplantation Institute,University of Pittsburgh Medical Center, StarzlBiomedical Science Tower, W1543, 200 LothropStreet, Pittsburgh, PA 15261, USA, (E-mail:cooperdk@upmc.edu)

*Contributed equally.

Received 11 December 2013;Accepted 20 January 2014

Introduction

Xenotransplantation could provide an unlimitedand elective supply of organs and cells for clinicaltransplantation. Although advances have beenmade with the introduction of a1,3-galactosyl-transferase gene-knockout (GTKO) pigs [1], evenafter the removal of this dominant xenoantigen,the anti-non-Gal antibody barrier still presents achallenge.

Specific pathogen-free (SPF) baboons wereestablished to reduce colonization with bacteria,viruses, fungi, and parasites that might be patho-genic. As it is believed that natural antibodies,

including some anti-pig antibodies, develop as aresult of microbial colonization of the intestinaltract during infancy [2–10], a reduced level of colo-nization might be associated with lower levels ofanti-pig antibody. We have investigated thishypothesis in baboons.

Methods

Baboons

Baboons (Papio species; SPF n = 8; non-SPFn = 32) were obtained from the Oklahoma Univer-sity Health Sciences Center (Oklahoma City, OK).

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Their age was 3–4 yr and weight was 6–9 kg.Baboons came from both conventional (non-SPF)and SPF colonies. The non-SPF colony is housedin indoor/outdoor pens, and the SPF colony ishoused in facilities that are all indoors. Breedinggroups in both colonies are similar in size andstructure (multimale and multifemale) with 40–80individuals in each group. The non-SPF colony isknown to harbor the normal endogenous viralpathogens present in baboon colonies (Table 1),including HVP1, HVP2, SVV, BaCMV, HHV6,BaRV, SFV, SRV, SIV, STLV, SV40, measles, andmonkeypox. However, these have been eliminatedfrom the SPF colony. Furthermore, the two inter-nal parasites, Strongyloides sp. and Trichuris trichi-ura, endemic in the non-SPF colony, have beeneliminated from the SPF colony.

Monitoring

Before the baboons had undergone any surgical orimmunomodulatory intervention, blood was col-lected by venepuncture for measurement of hema-tologic, biochemical, and coagulation parametersusing standard methods (Central Laboratory ofPresbyterian Hospital of the University of Pitts-burgh Medical Center, Pittsburgh, PA) [11].

Measurement of anti-non-Gal IgM and IgG by flow cytometry

Baboon serum samples were incubated for 30 minat 56 °C to inactivate complement. GTKO pig aor-tic endothelial cells were used as target cells. IgMand IgG antibodies directed to antigen targets

other than galactose-a1,3-galactose (anti-non-Galantibodies) were measured by immunofluorescenceintensity. Measurement of mean fluorescence in-tensity (MFI) was accomplished by CellQuest soft-ware (BD Biosciences, San Jose, CA, USA) usingLSR flow cytometry (San Jose, CA, USA) and rel-ative MFI was calculated by Flowjo software(Ashland, OR, USA).

Statistical analyses

The results were analyzed by Student’s t-test oranalysis of variance (ANOVA) where appropriate.The t-test was used to assess whether the mean val-ues of the SPF and non-SPF groups were statisti-cally different. A P value of < 0.05 was consideredto be statistically significant. Correlation of MFIwas calculated by linear regression analysis. Signif-icance at the 95% or the 99% level was calculatedusing prism-4 software (Graphpad Software, SanDiego, CA, USA).

Results

There were no significant differences in completeblood count or blood chemistry between SPF andnon-SPF baboons (Table 2). Anti-non-Gal IgMantibody levels were significantly lower in the SPFbaboons than in the non-SPF baboons (MFI 7.1vs. 8.8, P < 0.05) (Fig. 1). There was one SPFbaboon with a particularly high level of anti-non-Gal IgM (MFI 23.2) and two non-SPF baboonswith a MFI >20; if these three baboons are omittedfrom the calculations, the mean MFIs were 4.8(SPF) vs. 7.5 (non-SPF) (P < 0.05). Anti-non-GalIgG was minimal in both groups (MFI 1.0 vs. 1.0,NS) (Fig. 1).

Table 1. Viruses and parasites present in the non-SPF baboon colony

but eliminated from the SPF baboons

Herpesvirus

Herpesvirus papio 1 (HVP1)

Herpesvirus papio 2 (HVP2)Simian varicella zoster virus (SVV)

Baboon cytomegalovirus (BaCMV)

Human herpes virus 6 (HVP1)

Baboon rhadinovirus (BaRV)

Retrovirus

Simian foamy virus (SFV)

Simian retrovirus/D (SRV)

Simian immunodeficiency virus (SIV)

Simian T lymphotropic virus (STLV)

Papovavirus

Simian virus 40 (SV40)

Paramyxovirus

Morbillivirus (measles)

Orthopoxvirus

Monkeypox virus

Internal parasites

Trichuris trichuria (whipworms)

Stongyloides sp. (threadworms)

Table 2. Hematologic and blood chemistry parameters in SPF (n = 8)

and non-SPF (n = 32) baboons

SPF Non-SPF

Hematologic parameter (units)

WBC (103/ll) 6.32 6.58

RBC(106/ll) 5.16 4.97

Hct(%) 39.5 37.7

Hb(g/dl) 12.8 12.3

Plt(103/ll) 304 298

Blood chemistry parameters (units)

Alanine transaminase (IU/l) 35 34

Aspartate transaminase (IU/l) 30 28Gamma-glutamyl transferase (IU/l) 51 51

Blood urea nitrogen (mg/dl) 12 14

Creatinine (mg/dl) 0.6 0.7

Total protein (g/dl) 6.9 6.8

Albumin (g/dl) 4.1 3.9

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Discussion

Natural antibody levels, for example, anti-Galantibody, are believed to be associated with micro-bial colonization of the gastrointestinal tract of theanimal during the first few months of life [2–10].The development of antibody is generally consid-ered to be T cell-independent [12], although thereis some evidence that it may be T cell-dependent[13,14]. Although several factors, such as age, gen-der, ABO blood type, and vaccination history,may affect the levels of these antibodies [15], thelevel may be influenced by the extent of coloniza-tion of the animal. Baboons housed under SPFconditions, in which many of the usual microor-ganisms have been eliminated, might therefore beanticipated to express lower levels of natural anti-body. With regard to anti-non-Gal IgM, thisproved to be the case in the SPF baboons in thepresent study.

As anti-Gal antibody is no longer of significancein xenotransplantation studies because of the avail-ability of GTKO pigs, we did not measure anti-GalIgM or IgG levels. However, the mean level ofanti-non-Gal IgM (though not of IgG) was signifi-cantly lower in the SPF baboons than in the non-SPF baboons. Baboons maintained under SPFconditions may therefore be beneficial for xeno-transplantation studies as the initial binding ofanti-pig IgM to a GTKO pig organ may be less,thus resulting in less complement deposition and/or endothelial cell activation. However, even underidentical SPF conditions, an occasional baboonwill express a high level of anti-non-Gal IgM, thereason for which remains uncertain.

To our knowledge, there has been only one pre-vious study comparing antibody levels in baboonshoused under SPF or non-SPF conditions [16].Although there were differences in the assays(resulting in higher MFIs in the previous study),the results were similar to those reported here, withanti-non-Gal IgM being lower in SPF baboons,

with little difference in IgG. It may be significantthat the longest survivals to date of GTKO pigheart grafts have been reported in baboons thathad been housed under SPF conditions [16,17].

It is of interest to compare the levels of anti-non-Gal IgM and IgG in baboons with those inhumans [15,16]. The lower levels in SPF baboonsare closer to those in humans and, although thereare some variations in humans related to geo-graphic environment [15], this may reflect thereduced colonization of the gastrointestinal tract inSPF baboons and humans.

In the USA, the current cost of purchasing SPFbaboons is significantly higher than purchasingnon-SPF baboons (>$8500 vs. <$7000), but thisdiscrepancy is likely to be reduced in the future.The cost of establishing an SPF colony is high [18],but, once established, maintaining a baboon insuch a facility is minimally more than under con-ventional conditions, the only extra costs beingrelated to twice yearly testing for pathogens on theexclusion list. The purchase price should fall as thecolony reaches full maturity.

Acknowledgments

Burcin Ekser, MD, is a recipient of NIH NIAIDT32 AI 074490 training grant. Mohamed Ezzelarab,MD, is supported in part by the Joseph A. PatrickFellowship at the Thomas E. Starzl TransplantationInstitute. Work on xenotransplantation in theThomas E. Starzl Transplantation Institute of theUniversity of Pittsburgh is, or has been, supportedin part by NIH Grants #U19 AI090959, #U01AI068642, and # R21 A1074844 and by SponsoredResearch Agreements between the University ofPittsburgh and Revivicor, Inc., Blacksburg, VA.The baboons used in the study were from theOklahoma University Health Sciences Center,Baboon Research Resources, which is supported bythe Office Of The Director, NIH under AwardNumber P40OD010431 and P40OD010988.

Fig. 1. Anti-non-Gal IgM and IgG levelsin SPF (n = 8) and non-SPF (n = 32)baboons.

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Disclosure of conflict of interest

No author has a conflict of interest.

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