Early Feasibility Study of a Hybrid Tissue-Engineered Mitral Valve in an Ovine Model.

Tissue engineering aims to overcome the current limitations of heart valves by providing a viable alternative using living tissue. Nevertheless, the valves constructed from either decellularized xenogeneic or purely biologic scaffolds are unable to withstand the hemodynamic loads, particularly in the left ventricle. To address this, we have been developing a hybrid tissue-engineered heart valve (H-TEHV) concept consisting of a nondegradable elastomeric scaffold enclosed in a valve-like living tissue constructed from autologous cells. We developed a 21 mm mitral valve scaffold for implantation in an ovine model. Smooth muscle cells/fibroblasts and endothelial cells were extracted, isolated, and expanded from the animal's jugular vein. Next, the scaffold underwent a sequential coating with the sorted cells mixed with collagen type I. The resulting H-TEHV was then implanted into the mitral position of the same sheep through open-heart surgery. Echocardiography scans following the procedure revealed an acceptable valve performance, with no signs of regurgitation. The valve orifice area, measured by planimetry, was 2.9 cm2, the ejection fraction reached 67%, and the mean transmitral pressure gradient was measured at 8.39 mmHg. The animal successfully recovered from anesthesia and was transferred to the vivarium. Upon autopsy, the examination confirmed the integrity of the H-TEHV, with no evidence of tissue dehiscence. The preliminary results from the animal implantation suggest the feasibility of the H-TEHV.

Unique molecular signatures of antiviral memory CD8+ T cells associated with asymptomatic recurrent ocular herpes.

The nature of antiviral CD8+ T cells associated with protective and pathogenic herpes simplex virus type 1 (HSV-1) infections remains unclear. We compared the transcriptome, phenotype, and function of memory CD8+ T cells, sharing the same HSV-1 epitope-specificities, from infected HLA-A*0201 positive symptomatic (SYMP) vs. asymptomatic (ASYMP) individuals and HLA-A*0201 transgenic rabbits. Compared to higher frequencies of multifunctional effector memory CD8+ TEM cells in ASYMP individuals, the SYMP individuals presented dysfunctional CD8+ TEM cells, expressing major exhaustion pathways. Compared to protected ASYMP HLA transgenic rabbits, the trigeminal ganglia of non-protected SYMP HLA transgenic rabbits had higher frequencies of dysfunctional tissue-resident CD8+ TRM cells. Moreover, blockade of T cell exhaustion pathways restored the function of CD8+ T cells, reduced virus reactivation, and diminished recurrent disease in HLA transgenic rabbits. These findings reveal unique molecular signatures of protective CD8+ T cells and pave the way for T-cell-based immunotherapy to combat recurrent ocular herpes.

Blockade of PD-1 and LAG-3 Immune Checkpoints Combined with Vaccination Restores the Function of Antiviral Tissue-Resident CD8+ TRM Cells and Reduces Ocular Herpes Simplex Infection and Disease in HLA Transgenic Rabbits.

Chronic viruses such as herpes simplex virus 1 (HSV-1) evade the hosts' immune system by inducing the exhaustion of antiviral T cells. In the present study, we found that exhausted HSV-specific CD8+ T cells, with elevated expression of programmed death ligand-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) receptors were frequent in symptomatic patients, with a history of numerous episodes of recurrent corneal herpetic disease, compared to asymptomatic patients who never had corneal herpetic disease. Subsequently, using a rabbit model of recurrent ocular herpes, we found that the combined blockade of PD-1 and LAG-3 pathways with antagonist antibodies significantly restored the function of tissue-resident antiviral CD8+ TRM cells in both the cornea and the trigeminal ganglia (TG). An increased number of functional tissue-resident HSV-specific CD8+ TRM cells in latently infected rabbits was associated with protection against recurrent herpes infection and disease. Compared to the PD-1 or LAG-3 blockade alone, the combined blockade of PD-1 and LAG-3 appeared to have a synergistic effect in generating frequent polyfunctional Ki-67+, IFN-γ+, CD107+, and CD8+ T cells. Moreover, using the human leukocyte antigen (HLA) transgenic rabbit model, we found that dual blockade of PD-1 and LAG-3 reinforced the effect of a multiepitope vaccine in boosting the frequency of HSV-1-specific CD8+ TRM cells and reducing disease severity. Thus, both the PD-1 and the LAG-3 exhaustion pathways play a fundamental role in ocular herpes T cell immunopathology and provide important immune checkpoint targets to combat ocular herpes.IMPORTANCE HSV-specific tissue-resident memory CD8+ TRM cells play a critical role in preventing virus reactivation from latently infected TG and subsequent virus shedding in tears that trigger the recurrent corneal herpetic disease. In this report, we determined how the dual blockade of PD-1 and LAG-3 immune checkpoints, combined with vaccination, improved the function of CD8+ TRM cells associated with a significant reduction in recurrent ocular herpes in HLA transgenic (Tg) rabbit model. The combined blockade of PD-1 and LAG-3 appeared to have a synergistic effect in generating frequent polyfunctional CD8+ TRM cells that infiltrated both the cornea and the TG. The preclinical findings using the established HLA Tg rabbit model of recurrent herpes highlight that blocking immune checkpoints combined with a T cell-based vaccine would provide an important strategy to combat recurrent ocular herpes in the clinic.

Human Asymptomatic Epitope Peptide/CXCL10-Based Prime/Pull Vaccine Induces Herpes Simplex Virus-Specific Gamma Interferon-Positive CD107+ CD8+ T Cells That Infiltrate the Corneas and Trigeminal Ganglia of Humanized HLA Transgenic Rabbits and Protect against Ocular Herpes Challenge.

Herpes simplex virus 1 (HSV-1) is a prevalent human pathogen that infects the cornea, causing potentially blinding herpetic disease. A clinical herpes vaccine is still lacking. In the present study, a novel prime/pull vaccine was tested in a human leukocyte antigen (HLA) transgenic rabbit model of ocular herpes (HLA Tg rabbits). Three peptide epitopes were selected, from the HSV-1 membrane glycoprotein C (UL44400-408), the DNA replication binding helicase (UL9196-204), and the tegument protein (UL25572-580), all preferentially recognized by CD8+ T cells from "naturally protected" HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who never had recurrent corneal herpetic disease). HLA Tg rabbits were immunized with a mixture of these three ASYMP CD8+ T cell peptide epitopes (UL44400-408, UL9196-204, and UL25572-580), which were delivered subcutaneously with CpG2007 adjuvant (prime). Fifteen days later, half of the rabbits received a topical ocular treatment with a recombinant neurotropic adeno-associated virus type 8 (AAV8) vector expressing the T cell-attracting CXCL10 chemokine (pull). The frequency and function of HSV-specific CD8+ T cells induced by the prime/pull vaccine were assessed in the peripheral blood, cornea, and trigeminal ganglion (TG). Compared to the cells generated in response to peptide immunization alone, the peptide/CXCL10 prime/pull vaccine generated frequent polyfunctional gamma interferon-positive (IFN-γ+) CD107+ CD8+ T cells that infiltrated both the cornea and TG. CD8+ T cell mobilization into the cornea and TG of prime/pull-vaccinated rabbits was associated with a significant reduction in corneal herpesvirus infection and disease following an ocular HSV-1 (strain McKrae) challenge. These findings draw attention to the novel prime/pull vaccine strategy for mobilizing antiviral CD8+ T cells into tissues to protect against herpesvirus infection and disease.IMPORTANCE There is an urgent need for a vaccine against widespread herpes simplex virus infections. The present study demonstrates that immunization of HLA transgenic rabbits with a peptide/CXCL10 prime/pull vaccine triggered mobilization of HSV-specific CD8+ T cells locally into the cornea and TG, the sites of acute and latent herpesvirus infections, respectively. Mobilization of antiviral CD8+ T cells into the cornea and TG of rabbits that received the prime/pull vaccine was associated with protection against ocular herpesvirus infection and disease following an ocular HSV-1 challenge. These results highlight the importance of the prime/pull vaccine strategy to bolster the number and function of protective CD8+ T cells within infected tissues.

The Herpes Simplex Virus Latency-Associated Transcript Gene Is Associated with a Broader Repertoire of Virus-Specific Exhausted CD8+ T Cells Retained within the Trigeminal Ganglia of Latently Infected HLA Transgenic Rabbits.

UNLABELLED: Persistent pathogens, such as herpes simplex virus 1 (HSV-1), have evolved a variety of immune evasion strategies to avoid being detected and destroyed by the host's immune system. A dynamic cross talk appears to occur between the HSV-1 latency-associated transcript (LAT), the only viral gene that is abundantly transcribed during latency, and the CD8(+)T cells that reside in HSV-1 latently infected human and rabbit trigeminal ganglia (TG). The reactivation phenotype of TG that are latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT(+)TG) is significantly higher than TG latently infected with LAT-null mutant (i.e., LAT(-)TG). Whether LAT promotes virus reactivation by selectively shaping a unique repertoire of HSV-specific CD8(+)T cells in LAT(+)TG is unknown. In the present study, we assessed the frequency, function, and exhaustion status of TG-resident CD8(+)T cells specific to 40 epitopes derived from HSV-1 gB, gD, VP11/12, and VP13/14 proteins, in human leukocyte antigen (HLA-A*0201) transgenic rabbits infected ocularly with LAT(+)versus LAT(-)virus. Compared to CD8(+)T cells from LAT(-)TG, CD8(+)T cells from LAT(+)TG (i) recognized a broader selection of nonoverlapping HSV-1 epitopes, (ii) expressed higher levels of PD-1, TIM-3, and CTLA-4 markers of exhaustion, and (iii) produced less tumor necrosis factor alpha, gamma interferon, and granzyme B. These results suggest a novel immune evasion mechanism by which the HSV-1 LAT may contribute to the shaping of a broader repertoire of exhausted HSV-specific CD8(+)T cells in latently infected TG, thus allowing for increased viral reactivation. IMPORTANCE: A significantly larger repertoire of dysfunctional (exhausted) HSV-specific CD8(+)T cells were found in the TG of HLA transgenic rabbits latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT(+)TG) than in a more restricted repertoire of functional HSV-specific CD8(+)T cells in the TG of HLA transgenic rabbits latently infected with LAT-null mutant (i.e., LAT(-)TG). These findings suggest that the HSV-1 LAT locus interferes with the host cellular immune response by shaping a broader repertoire of exhausted HSV-specific CD8(+)T cells within the latency/reactivation TG site.

Large Amounts of Reactivated Virus in Tears Precedes Recurrent Herpes Stromal Keratitis in Stressed Rabbits Latently Infected with Herpes Simplex Virus.

AIM: Recurrent herpetic stromal keratitis (rHSK), due to an immune response to reactivation of herpes simplex virus (HSV-1), can cause corneal blindness. The development of therapeutic interventions such as drugs and vaccines to decrease rHSK have been hampered by the lack of a small and reliable animal model in which rHSK occurs at a high frequency during HSV-1 latency. The aim of this study is to develop a rabbit model of rHSK in which stress from elevated temperatures increases the frequency of HSV-1 reactivations and rHSK. MATERIALS AND METHODS: Rabbits latently infected with HSV-1 were subjected to elevated temperatures and the frequency of viral reactivations and rHSK were determined. RESULTS: In an experiment in which rabbits latently infected with HSV-1 were subjected to ill-defined stress as a result of failure of the vivarium air conditioning system, reactivation of HSV-1 occurred at over twice the normal frequency. In addition, 60% of eyes developed severe rHSK compared to <1% of eyes normally. All episodes of rHSK were preceded four to five days prior by an unusually large amount of reactivated virus in the tears of that eye and whenever this unusually large amount of reactivated virus was detected in tears, rHSK always appeared 4-5 days later. In subsequent experiments using well defined heat stress the reactivation frequency was similarly increased, but no eyes developed rHSK. CONCLUSIONS: The results reported here support the hypothesis that rHSK is associated not simply with elevated reactivation frequency, but rather with rare episodes of very high levels of reactivated virus in tears 4-5 days earlier.

Effect of Room Ventilation Rates in Rodent Rooms with Direct-Exhaust IVC Systems.

When IVC are directly exhausted from a rodent housing room, the air quality of the room can become independent of the intracage air quality and may reduce the need for high room ventilation rates. This study assessed the effect of decreasing the ventilation rate in rodent rooms using direct-exhaust IVC systems. The study was conducted over 16 wk and compared conditions in 8 rodent rooms that had ventilation rates of 5 to 6 air changes per hour (ACH) with those in rooms at 10 to 12 ACH. At the low ventilation rate, rooms had higher CO2 concentrations, higher dew point temperature, and lower particulate levels and spent a greater percentage of time above the temperature set point than did rooms at the high rate. The levels of allergens and endotoxins in room air were the same regardless of the ventilation rate. Differences seen in parameters within cages at the 2 ventilation rates were operationally irrelevant. We detected no total volatile organic compounds in the room that were attributable to ammonia, regardless of the ventilation rate. Clearing the air of ethanol after a spill took longer at the low compared with high rate. However, ethanol clearance was faster at the low rate when the demand-control system was activated than at the high ventilation rate alone. Air quality in the room and in the cages were acceptable with room ventilation rates of 5 to 6 ACH in rodent rooms that use direct-exhaust IVC systems.

Therapeutic immunization with a mixture of herpes simplex virus 1 glycoprotein D-derived "asymptomatic" human CD8+ T-cell epitopes decreases spontaneous ocular shedding in latently infected HLA transgenic rabbits: association with low frequency of local PD-1+ TIM-3+ CD8+ exhausted T cells.

UNLABELLED: Most blinding ocular herpetic disease is due to reactivation of herpes simplex virus 1 (HSV-1) from latency rather than to primary acute infection. No herpes simplex vaccine is currently available for use in humans. In this study, we used the HLA-A*02:01 transgenic (HLA Tg) rabbit model of ocular herpes to assess the efficacy of a therapeutic vaccine based on HSV-1 gD epitopes that are recognized mainly by CD8(+) T cells from "naturally" protected HLA-A*02:01-positive, HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease). Three ASYMP CD8(+) T-cell epitopes (gD(53-61), gD(70-78), and gD(278-286)) were linked with a promiscuous CD4(+) T-cell epitope (gD(287-317)) to create 3 separate pairs of CD4-CD8 peptides, which were then each covalently coupled to an Nε-palmitoyl-lysine moiety, a Toll-like receptor 2 (TLR-2) ligand. This resulted in the construction of 3 CD4-CD8 lipopeptide vaccines. Latently infected HLA Tg rabbits were immunized with a mixture of these 3 ASYMP lipopeptide vaccines, delivered as eye drops in sterile phosphate-buffered saline (PBS). The ASYMP therapeutic vaccination (i) induced HSV-specific CD8(+) T cells that prevent HSV-1 reactivation ex vivo from latently infected explanted trigeminal ganglia (TG), (ii) significantly reduced HSV-1 shedding detected in tears, (iii) boosted the number and function of HSV-1 gD epitope-specific CD8(+) T cells in draining lymph nodes (DLN), conjunctiva, and TG, and (iv) was associated with fewer exhausted HSV-1 gD-specific PD-1(+) TIM-3+ CD8(+) T cells. The results underscore the potential of an ASYMP CD8(+) T-cell epitope-based therapeutic vaccine strategy against recurrent ocular herpes. IMPORTANCE: Seventy percent to 90% of adults harbor herpes simplex virus 1 (HSV-1), which establishes lifelong latency in sensory neurons of the trigeminal ganglia. This latent state sporadically switches to spontaneous reactivation, resulting in viral shedding in tears. Most blinding herpetic disease in humans is due to reactivation of HSV-1 from latency rather than to primary acute infection. To date, there is no licensed therapeutic vaccine that can effectively stop or reduce HSV-1 reactivation from latently infected sensory ganglia and the subsequent shedding in tears. In the present study, we demonstrated that topical ocular therapeutic vaccination of latently infected HLA transgenic rabbits with a lipopeptide vaccine that contains exclusively human "asymptomatic" CD8(+) T-cell epitopes successfully decreased spontaneous HSV-1 reactivation, as judged by a significant reduction in spontaneous shedding in tears. The findings should guide the clinical development of a safe and effective T-cell-based therapeutic herpes vaccine.

Protection of mice against H. somni septicemia by vaccination with recombinant immunoglobulin binding protein subunits.

Histophilus somni causes bovine pneumonia as well as septicemia and its sequelae but mechanisms of virulence and protective immunity are poorly understood. Since surface immunoglobulin binding proteins are virulence factors, we addressed their role as protective antigens in a mouse model of H. somni septicemia. Immunoglobulin binding protein A (IbpA), has homology to Bordetella pertussis filamentous hemagglutinin and other large bacterial exoproteins. IbpA is a major surface antigen encoded by the ibpA gene with many domains that may be important in pathogenesis and immune protection. Three IbpA recombinant protein subunits, IbpA3, IbpA5 and IbpADR2 were chosen for study because of putative functional domains and motifs. These recombinant GST fusion subunit proteins were compared with GST (negative control), formalin-killed H. somni (commercial vaccine control), live H. somni (to induce convalescent immunity) and H. somni culture supernatant (containing IbpA shed from the bacterial surface). In vaccination/challenge studies, both live H. somni (convalescent immunity) and supernatant protected equally but formalin-killed H. somni and GST did not protect against septicemia. The DR2 and A3 subunits protected moderately well and induced antibody responses against supernatant antigen and the homologous subunit in ELISA but not against whole cell antigens. Supernatant immunization protected better than the IbpA subunit antigens and induced high antibody activity against both whole cells and supernatant antigens. The results indicate that culture supernatant antigens or perhaps recombinant IbpA subunits may be useful in H. somni vaccines. These studies also provide insight into the contribution of IbpA domains to pathogenesis of H. somni septicemia.

Bovine plasma proteins increase virulence of Haemophilus somnus in mice.

The role of bovine serum or plasma proteins in Haemophilus somnus virulence was investigated in a mouse model of septicemia. An increase in virulence was detected when the organism was pre-incubated for 5 min and inoculated with fetal calf serum. When purified bovine serum or plasma proteins were pre-incubated with H. somnus before inoculating into mice, transferrin was found to increase virulence. Bovine lactoferrin was also noted to increase virulence, but to a lesser extent and had a delayed time course when compared with transferrin. Using an ELISA assay, an increased amount of H. somnus whole cells and culture supernatant bound to bovine transferrin when the organism was grown in iron-restricted media. Lactoferrin also bound to H. somnus, but binding was not affected by growth in iron-restricted media and it was eliminated with 2M NaCl, which reversed charge mediated binding. Transferrin, but not lactoferrin, supported growth of H. somnus on iron-depleted agar based media using a disk assay. Therefore, lactoferrin increased virulence by an undetermined mechanism whereas transferrin increased virulence of H. somnus by binding to iron-regulated outer-membrane proteins (IROMPs) and providing iron to the pathogen.