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. 2015 Jul;15(7):1805-16.
doi: 10.1111/ajt.13188. Epub 2015 Mar 12.

Rat Cytomegalovirus Vaccine Prevents Accelerated Chronic Rejection in CMV-Naïve Recipients of Infected Donor Allograft Hearts

D N Streblow  1   2 , Y K Hwee  3 , C N Kreklywich  2   3 , T Andoh  3   4 , M Denton  2 , P Smith  2 , E Hart  2 , R Broekel  2 , C Pallett  2 , K Rogers  2 , A D Streblow  2 , M Chuop  2 , A Perry  1 , M Slifka  1   5 , I Messaoudi  6 , S L Orloff  1   3   4
Affiliations

Rat Cytomegalovirus Vaccine Prevents Accelerated Chronic Rejection in CMV-Naïve Recipients of Infected Donor Allograft Hearts

D N Streblow et al. Am J Transplant. 2015 Jul.

Abstract

Cytomegalovirus accelerates transplant vascular sclerosis (TVS) and chronic rejection (CR) in solid organ transplants; however, the mechanisms involved are unclear. We determined the efficacy of a CMV vaccine in preventing CMV-accelerated rat cardiac allograft rejection in naïve recipients of CMV+ donor hearts. F344 donor rats were infected with RCMV 5 days prior to heterotopic cardiac transplantation into CMV-naïve or H2 O2 -inactivated RCMV-vaccinated Lewis recipients. Recipients of RCMV-infected donor hearts rejected at POD59, whereas vaccinated recipients exhibited a significantly prolonged time to rejection-POD97, similar to recipients of uninfected donor hearts (POD108). Although all of the donor hearts were preinfected, the vaccinated recipients had lower graft and PBMC viral loads at POD 7 compared to unvaccinated controls. Adoptive T cell and passive antibody transfers from vaccinated Lewis rats into naïve recipients demonstrate that both T-cell and B-cell arms of the adaptive immune response provide protection against CMV-accelerated rejection. Similar findings were obtained when testing three different adjuvants in passive transfer experiments. We have determined that the timing of the vaccine prior to transplantation and the specific adjuvant play critical roles in mediating anti-viral responses and promoting graft survival. CMV vaccination prior to transplantation may effectively increase graft survival.

Keywords: animal models; basic (laboratory) research / science; graft survival; heart transplantation / cardiology; infection and infectious agents; infectious disease; vaccine; viral: Cytomegalovirus (CMV).

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Figures

Figure 1
Figure 1
Vaccine experimental design. Depicted is the vaccine experimental strategy and study time line. A total of five groups were included in this study: (1) nonvaccinated recipient of an infected donor heart; (2) nonvaccinated recipient of uninfected donor heart; (3) vaccinated recipient at 35 days before transplantation that was boosted at 28 days later received an infected donor heart; (4) vaccinated recipient at 35 days before transplantation that was boosted 28 days later received an uninfected donor heart; (5) vaccinated recipient at 70 days before transplantation that was boosted 28 days later received an infected donor heart. Donors were infected at 5 days pretransplant to ensure that each donor heart was infected and that infection was not affected by pre‐existing immunity in the vaccinated groups, which would skew the results. Rejection was monitored and the animals were euthanized at the time of CR at which time blood, salivary glands, graft heart, and other major organs were harvested for analysis. A second cohort of Nonvaccinated, Day 35, and Day 70 vaccinated allograft recipients was harvested at postoperative day (POD) 7 to monitor the effect of vaccination on virus levels at early times posttransplant.
Figure 2
Figure 2
Vaccine promotes allograft survival of RCMV infected donor hearts. Vaccination of recipients with H2O2‐inactivated RCMV at 70 pretransplantation promotes survival of RCMV‐infected donor hearts compared to unvaccinated controls and recipients vaccinated at 35 days pretransplant. Heterotopic heart allografts (RCMV + / −) were transplanted into RCMV‐naïve recipients or animals vaccinated at 35 or 70 pretransplant (n = 6). Heart graft recipients were monitored over a 120‐day period for clinical graft rejection. (A) Graft survival curves were produced using GraphPad Prism 5 software. (B) Graph depicts the mean time of rejection for each group as well as the time to rejection for each replicate. (C) Sections of graft hearts were stained with H&E and elastic von Gieson in order to determine the degree of vascular disease in the coronary arteries. The average neointimal index (vascular disease, TVS) for each group as well as the NI for each individual heart graft were graphed (means and SEM) using Prism 5 software. Standard deviation is indicated with error bars. The severity of TVS (NI scores) between the vaccine groups was analyzed and compared using Student's t‐test (p‐values < 0.05 were considered statistically significant).
Figure 3
Figure 3
Day 70 vaccine reduced viral load in allograft recipients at CR. Total DNA was prepared using the DNAzol method. Viral DNA loads were determined by quantitative PCR using virus specific primers and probe; and then the results were compared between the groups using Student's t‐test (p‐values < 0.05 were considered statistically significant). (A) Vaccination of recipients with H2O2‐inactivated RCMV at 70 days pretransplant reduces viral load in salivary glands at the time of graft rejection compared to unvaccinated controls and recipients vaccinated with 35‐day regimen (*p < 0.01). (B) Vaccination of recipients with H2O2‐inactivated RCMV at POD −70 significantly reduces viral load in recipient PBMC and allograft heart but not salivary glands at 7 days posttransplantation compared to unvaccinated controls and recipients that were vaccinated at POD −35. SMG: submandibular gland.
Figure 4
Figure 4
Vaccine elicits increased antibody responses to RCMV. The production of anti‐RCMV antibodies in the sera from vaccinated recipients at various times pre‐ and posttransplantation was measured using standard ELISA detecting RCMV‐infected cell lysates as the antigenic source. (A) Total IgG antibody binding was detected using a pan‐anti rat IgG antibody coupled to horseradish peroxidase. CR = chronic rejection. (B) Primary antibody binding was detected using anti‐rat isotype‐specific secondary antibodies (IgG1, IgG2a, IgG2b, IgG2c, and IgM). Animals vaccinated against inactivated RCMV with Freund's adjuvant using the 70‐day vaccine regimen were higher for IgM, IgG2b and IgG2c compared to animals vaccinated using the 35‐day vaccine protocol. N = 6 per group. (C) Western blot analysis was used to measure the presence and breadth of the antibody response in serum from uninfected control rats, the 35 day and 70 day vaccine groups compared to a rat monoclonal antibody that recognizes gB. RCMV infected cell lysates were separated by SDS‐PAGE and transferred to PVDF membrane. Blots were blocked and primary antibodies were bound for 1 h at room temperature. Primary antibody binding was detected using anti‐rat total Ig conjugated to horseradish peroxidase and developed by ECL and autoradiography.
Figure 5
Figure 5
Day 70 vaccine is more efficacious at promoting neutralizing antibody development in transplanted rats. Plaque reduction neutralizing assays were performed on serum from nonvaccinated controls, Day 35 vaccine and Day 70 vaccine groups; (n = 6). A 1:20 dilution of serum was added to 100 pfu of RCMV for 1 h at 37°C and then added to rat fibroblasts for plaque assay. Data is presented as percent plaque reduction compared to control sera.
Figure 6
Figure 6
Identifying correlates of protection for anti‐RCMV vaccine. To determine the mechanisms that mediate protection from RCMV infection/accelerated rejection in the vaccinated animals, we performed passive transfer of either serum or splenic T cells from vaccinated rats into RCMV‐naïve recipients prior to transplantation of an infected donor heart. (A) Depicted is the vaccine/transfer experimental strategy. Naïve Lewis rats were vaccinated with hydrogen peroxide inactivated RCMV using the 70‐day regimen. At 1 day pretransplant, CMV‐naïve Lewis recipient rats were injected with 1 mL of serum or a mixture of 6 × 106 CD4+ and CD8+ central memory or effector memory T cells isolated from total splenocytes by flow cytometry activated cell sorting FACS (n = 6). Shown is the analysis of the rat CD4/CD8 effector and central memory cells. The recipient rats were transplanted with an infected F344 donor heart. Recipients of uninfected rats served as negative controls and recipients of infected hearts (untreated) served as positive controls for RCMV‐accelerated CR. Time to rejection was determined for each group and graphed in a survival plot (B) or graphed as the average time to rejection (C). Shown in panel D is the average neointimal index of allograft vessels at the time of chronic rejection. Transfer of immune serum prevented RCMV‐accelerated rejection and TVS formation in infected donor hearts compared to controls.
Figure 7
Figure 7
Transfer of serum or T cells from vaccinated rats to naïve recipients reduced viral load in allograft recipient salivary glands at CR. Transfer of vaccine immune serum or T cells reduces viral load in salivary glands at the time of graft rejection compared to unvaccinated controls (p < 0.02). Total DNA was prepared using DNAzol and viral genomic DNA levels were measured by quantitative PCR. The results were compared between the groups using Student's t‐test.
Figure 8
Figure 8
Anti‐RCMV vaccine efficacy is adjuvant‐dependent. Three adjuvants were compared for their effect on efficacy of the anti‐RCMV vaccine in recipients of CMV‐infected donor allografts. CMV‐naïve Lewis rats were vaccinated with hydrogen peroxide inactivated RCMV adjuvanted with Alum, MPL, Alum + MPL, or Freund's using the 70‐day vaccination regimen. At 1 day pretransplant, Lewis recipient rats were injected with 1 mL of blood serum derived from the vaccinated animals (n = 6). The recipient rats were transplanted with a RCMV‐infected F344 donor heart. Group 1 received serum from vaccinated animals with H202‐inactivated virus alone with no added adjuvant; Group 2 vaccine was adjuvanted with Alum; Group 3 with MPL; Group 4 with Alum + MPL; Group 5 with Freund's adjuvant; Group 6 were untreated recipients of infected hearts to serve as positive controls for RCMV‐accelerated CR; and Group 7 were treated with a rat monoclonal antibody directed against RCMV‐gB that neutralizes in the presence of complement. (A) Time to rejection was determined for each group and graphed in a survival plot (B) or graphed as the average time to rejection. Shown in panel C is the average neointimal index of allograft vessels at the time of chronic rejection. Transfer of immune serum from rats vaccinated using the adjuvant Alum + MPL prevented RCMV‐accelerated rejection and TVS formation in infected donor hearts to levels observed for those animals receiving Freund's adjuvant.
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References

    1. Bruning JH, Persoons MCJ, Lemstrom KB, Stals FS, De Clereq E, Bruggeman CA. Enhancement of transplantation associated atherosclerosis by CMV, which can be prevented by antiviral therapy in the form of HPMPC. Transplant Int 1994; 7: 365–370. - PubMed
    1. Lemstrom K, Koskinen P, Krogerus L, Daemen M, Bruggeman CA, Hayry PJ. Cytomegalovirus antigen expression, endothelial cell proliferation, and intimal thickening in rat cardiac allografts after cytomegalovirus infection. Circ 1995; 92: 2594–2604. - PubMed
    1. Lemstrom KB, Bruning JH, Bruggeman CA, Lautenschlager IT, Hayry PJ. Cytomegalovirus Infection Enhances Smooth Muscle Cell Proliferation and Intimal Thickening of Rat Aortic Allografts. J Clin Invest 1993; 92: 549–558. - PMC - PubMed
    1. Orloff SL. Elimination of donor‐specific alloreactivity by bone marrow chimerism prevents cytomegalovirus accelerated transplant vascular sclerosis in rat small bowel transplants. J Clin Virol 1999; 12: 142 (G3–25).
    1. Orloff SL, Streblow DN, Soderberg‐Naucler C, et al. Elimination of donor‐specific alloreactivity prevents cytomegalovirus‐accelerated chronic rejection in rat small bowel and heart transplants. Transplantation 2002; 73: 679–688. - PubMed

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