Outcomes of open reduction and internal fixation of calcaneus fractures: A database study comparing patients with and without diabetes.

Treatment of calcaneal fractures in patients with diabetes mellitus (DM) is challenging. The purpose of this study was to compare post-operative outcomes after open reduction and internal fixation (ORIF) for calcaneus fracture in patients with complicated DM, uncomplicated DM, and patients without DM. A commercially available de-identified database was queried for all calcaneus fracture diagnoses undergoing ORIF from 2010 to 2021. The patients were separated into three groups for analysis: patients without DM (10,951, 82.6%), uncomplicated DM (1,500, 11.3%) and complicated DM (802, 6.1%). At 1 year, post-operative adverse events were assessed among the three groups. The odds of adverse event(s) for each group were compared between the three groups with and without characteristic matching. In the unmatched cohorts, patients with complicated DM, when compared with patients without DM and patients with uncomplicated DM, had significantly higher rates of all adverse events with exception of DVT. Rates of CNA were significantly higher in patients with complicated DM compared with no DM (OR 107.7 (CI 24.83-467.6) p < 0.0001) and uncomplicated DM (OR 44.26 (CI 3.86-507.93) p = 0.0002). After matching, non-union, AKI, sepsis, surgical site infection, and wound disruption were higher in patients with complicated DM compared with patients without DM. There were no significant differences in the three groups with regard to reoperation, DVT, MI, pneumonia, or below the knee amputation. Patients with DM who underwent ORIF for calcaneus fracture experienced higher rates of post-operative adverse events compared with those patients without DM.

Cas9-induced targeted integration of large DNA payloads in primary human T cells via homology-mediated end-joining DNA repair.

The reliance on viral vectors for the production of genetically engineered immune cells for adoptive cellular therapies remains a translational bottleneck. Here we report a method leveraging the DNA repair pathway homology-mediated end joining, as well as optimized reagent composition and delivery, for the Cas9-induced targeted integration of large DNA payloads into primary human T cells with low toxicity and at efficiencies nearing those of viral vectors (targeted knock-in of 1-6.7 kb payloads at rates of up to 70% at multiple targeted genomic loci and with cell viabilities of over 80%). We used the method to produce T cells with an engineered T-cell receptor or a chimaeric antigen receptor and show that the cells maintained low levels of exhaustion markers and excellent capacities for proliferation and cytokine production and that they elicited potent antitumour cytotoxicity in vitro and in mice. The method is readily adaptable to current good manufacturing practices and scale-up processes, and hence may be used as an alternative to viral vectors for the production of genetically engineered T cells for cancer immunotherapies.

Fractures and dislocations of the foot and ankle in people with diabetes: a literature review.

Diabetes (DM) increases fracture risk, and bone quality depends on type diabetes type, duration, and other comorbidities. Diabetes is associated with a 32% increased relative risk (RR) of total fractures and 24% increased RR of ankle fractures compared with patients without DM. Type 2 DM is associated with a 37% increased RR of foot fractures compared with patients without DM. The incidence of ankle fractures in the general population is 169/100,000 per year, while foot fractures occur less frequently, with an incidence of 142/100,000 per year. Biomechanical properties of bone are negatively impacted by stiff collagen, contributing to the increased risk of fragility fractures in patients with DM. Systemic elevation of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNFα), interleukin-1ß (IL-1ß), and interleukin 6 (IL-6), impact bone healing in patients with DM. Fractures in patients with DM, can be associated with poorly regulated levels of RANKL (receptor activator of nuclear transcription factor kappa-b ligand) leading to prolonged osteoclastogenesis, and net bone resorption. One of the most salient factors in treating fractures and dislocations of the foot and ankle is to recognize the difference between patients with uncomplicated and complicated DM. Complicated diabetes is defined as 'end organ damage', and for the purposes of this review, includes patients with neuropathy, peripheral artery disease (PAD) and/or chronic renal disease. Uncomplicated diabetes is not associated with 'end organ damage'. Foot and ankle fractures in patients with complicated DM pose challenges, and surgery is associated with increased risks of impaired wound healing, delayed fracture healing, malunion, infection, surgical site infection, and revision surgery. While patients with uncomplicated DM can be treated like patients without DM, patients with complicated DM require close follow-up and robust fixation methods should be considered to withstand the anticipated prolonged healing period. The aims of this review are as follows: (1) to review pertinent aspects of DM bone physiology and fracture healing, (2) to review the recent literature on treatment of foot and ankle fractures in patients with complicated DM, and (3) to provide treatment protocols based on the recent published evidence.

Ankle Joint Salvage for Rigid Flatfoot Deformity.

Rigid flatfoot deformity with valgus ankle instability is a complex condition to treat. Thorough clinical and radiographic evaluation is vital to determine treatment strategies. Nonoperative treatment usually relies on bracing or various orthoses. Surgical interventions include ligament reconstruction, osteotomies, arthrodesis, arthroplasty, or a combination of these procedures. Before addressing the ankle deformity, a plantigrade foot is important so a staged approach may be necessary. Misalignment of the ankle replacement can lead to edge loading and early failure. As the implants and our understanding of ankle arthroplasty improve, more patients may benefit from a motion-preserving procedure rather than an arthrodesis.

Limb Salvage in Severe Diabetic Foot Infection.

Severe diabetic foot infections (DFI) are both limb threatening and life threatening and associated with negative impact on health-related quality of life. Most severe DFIs require surgical intervention, and the goal of treatment should be preservation of limb function in addition to eradication of infection. Minor amputations are required in approximately 40% and major amputations in approximately 20% of patients. Significant risk factors for lower extremity amputation included male gender, smoking, previous amputation, osteomyelitis, peripheral artery disease, retinopathy, severe infections, gangrene, neuroischemic diabetic foot infections, leukocytosis, positive wound cultures, and isolation of gram-negative bacteria.

A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes.

Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies.

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade.

BACKGROUND: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade. METHODS: Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade. FINDINGS: CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo. CONCLUSIONS: CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy. FUNDING: This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

A Systematic Review of Intramedullary Fixation in Midfoot Charcot Neuroarthropathy.

Charcot neuroarthropathy can cause severe deformity of the midfoot, and intramedullary use of beams and bolts has been utilized as a method of definitive stabilization. This systematic review evaluated the outcomes of intramedullary beaming in patients with Charcot neuroarthropathy and determined the methodological quality of the studies. Four online databases were searched: PubMed, MEDLINE (Clarivate Analytics), CINAHL (Cumulative Index to Nursing and Allied Health) and Web of Science (Clarivate Analytics). To assess the methodological quality of the studies, the Coleman Methodology Score was used. The data was pooled into 2 outcomes groups for comparison: (1) Studies that reported on the outcomes of Charcot specific implants (study group). (2) Studies that reported on the outcomes using non-Charcot specific implants (control group). After screening, 16 studies were included. Compared to our control group, our study group had significantly higher rates of overall hardware complications, hardware migration, surgical site infection, reoperation, and nonunion. The study group had significantly lower rates of limb salvage compared to the control group. Our study and control groups did not differ in the rates of hardware breakage, wound healing complications, or mortality. The limb salvage rate was 92% and 97% of patients were still alive at a mean follow-up of 25 months. The mean Coleman Methodology Score indicated the quality of the studies was poor and consistent with methodologic limitations. The quality of published studies on intramedullary implants for Charcot reconstruction is low. Complications when utilizing intramedullary fixation for Charcot reconstruction are high, whether or not Charcot specific implants are used.

Arthroscopic Findings in Refractory Symptomatic Fourth and Fifth Tarsometatarsal Joints.

Refractory pain to the fourth and fifth tarsometatarsal (TMT) joint can be a source of disability and functional impairment. While pain has been attributed to injury, post-traumatic arthritis, arthrofibrosis, the principal causes of pain in the absence of arthritis are not well elucidated. The purpose of this study is to characterize arthroscopic pathology associated with chronic refractory pain to the fourth and fifth TMT joints. We retrospectively examined 24 patients that underwent arthroscopic surgery of the fourth and fifth TMT joints for refractory pain at our academic institution between 2015 and 2019. We used the Outerbridge classification for chondral lesions, the Kellgren Lawrence radiographic classification for osteoarthritis, and described intraarticular pathologies as acute hypertrophic synovitis, chronic synovial fibrosis, hyaline bands, meniscoid bodies, loose joint bodies, arthrofibrosis. Approximately, 31 of 45 TMT joints (68.9%) presented with radiographic evidence of arthritis. Approximately, 14 of 45 TMT joints (31.11%) were absent of radiographic signs of arthritis. The frequency of soft tissue pathology seen in these patients without radiographic evidence of arthritis was arthrofibrosis (87.5%), chronic synovial fibrosis (75.0%), and acute hypertrophic synovitis (62.5%). This is the first study to report arthroscopic pathologies associated with refractory pain to the fourth and fifth TMT joints.

Identifying Risk Factors for Nonunion of the Modified Lapidus Procedure for the Correction of Hallux Valgus.

There is a paucity of literature characterizing risk factors for nonunion associated with the modified Lapidus procedure for correction of hallux valgus. The purpose of this study was to evaluate risk factors associated with nonunion for Lapidus bunionectomies. Patients who underwent modified Lapidus procedure from 2009 to 2018 were retrospectively reviewed. Patient's age, sex, body mass index, prior bunionectomy, history of tobacco use, presence of diabetes mellitus or hypothyroidism, and fixation method were recorded along with pre- and postoperative radiographic parameters. A multiple logistic regression analysis was implemented to estimate the odds of nonunion. Of the 222 patients who met inclusion criteria, nonunion with modified Lapidus procedure was observed in 20 patients (9.01%). Odds of nonunion with modified Lapidus procedure were greater for patients who had undergone previous bunionectomy (odds ratio [OR] = 3.957, 95% confidence interval [CI]: 1.021-15.338), as body mass index increased (OR = 1.091, 95% CI: 1.018-1.170), and as preoperative HV angle increased (OR = 1.108, 95% CI: 1.020-1.203). Odds of nonunion were lower for patients as preoperative intermetatarsal angle increased (OR = 0.739, 95% CI: 0.580-0.941). No significant increased odds of nonunion were found between fixation methods.

The Impact of Hospitalization for Diabetic Foot Infection on Health-Related Quality of Life: Utilizing PROMIS.

Diabetic foot infections (DFI) are an increasingly common cause of hospitalizations. Once hospitalized with DFI, many patients require some level of amputation, often undergoing multiple operations. With increasing importance on patient-centered metrics, self-reported health-related quality of life (HRQOL) tools have been developed. This prospective cohort study aimed assessed the impact of DFI on HRQOL. Two hundred twenty-four patients completed the 29-item Patient-Reported Outcome Measurement Information System (PROMIS) and 12-Item Short Form (SF-12) survey. Secondary outcomes using the Foot and Ankle Ability Measures survey were obtained and included in the analysis. The study group was comprised of hospitalized patients with DFIs (n = 120), and the control group was comprised of patients with diabetes who were evaluated for routine outpatient foot care (n = 104); diabetic foot screening, wound care, onychomycosis, and/or callosities. Using this cohort, a propensity score-matched sample of hospitalized patients with DFI (n = 35) and control group patients (n = 35) was created for comparative analysis. The 2-independent sample t test was used to test for group differences on each of the PROMIS subscale outcomes. Using PROMIS, we found that hospitalized patients with DFI reported significantly worse HRQOL in 6 of 7 subscales (physical function, anxiety, depression, fatigue, social role, pain intensity; p value range: .0001-.02) compared to outpatients with diabetes evaluated for routine foot care. There was no significant difference between the 2 groups on sleep disturbance (p = .22). Patients hospitalized for DFI report lower HRQOL compared to patients with diabetes receiving routine outpatient foot care.

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9.

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function-to produce large amounts of antibodies-can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.

The fusion of genome engineering and adoptive cellular therapy holds immense promise for the treatment of genetic disease and cancer. Multiplex genome engineering using targeted nucleases can be used to increase the efficacy and broaden the application of such therapies but carries safety risks associated with unintended genomic alterations and genotoxicity. Here, we apply base editor technology for multiplex gene modification in primary human T cells in support of an allogeneic CAR-T platform and demonstrate that base editor can mediate highly efficient multiplex gene disruption with minimal double-strand break induction. Importantly, multiplex base edited T cells exhibit improved expansion and lack double strand break-induced translocations observed in T cells edited with Cas9 nuclease. Our findings highlight base editor as a powerful platform for genetic modification of therapeutically relevant primary cell types.

Outcomes of Foot Infections Secondary to Puncture Injuries in Patients With and Without Diabetes.

It is difficult to compare foot infections in patients with diabetes to those without diabetes because foot infections are uncommon in people without diabetes. The aim of this study is to compare clinical outcomes in people with and without diabetes admitted to the hospital for an infected puncture wound. We evaluated 114 consecutive patients from June 2011 to March 2019 with foot infection resulting from a puncture injury; 83 had diabetes and 31 did not have diabetes. We evaluated peripheral arterial disease (PAD), sensory neuropathy, the need for surgery and amputation, length of hospitalization, and presence of osteomyelitis. Patients with diabetes were 31 times more likely to have neuropathy (91.6% versus 25.8%, p < .001, confidence interval [CI] 10.2 to 95.3), 8 times more likely to have PAD (34.9% versus 6.5%, p = .002, CI 1.7 to 35), and 7 times more likely to have kidney disease (19.3% versus 3.2%, p < .05, CI 0.9 to 56.5). They also took longer before presenting to the hospital (mean 20.1 ± 36.3 versus 18.8 ± 34.8 days, p = .09, CI 13 to 26.5); however, this result was not statistically significant. Patients with diabetes were 9 times more likely to have osteomyelitis (37.3% versus 6.5%, p = .001, CI 1.9 to 38.8). In addition, they were more likely to require surgery (95% versus 77%, p < .001, CI 1.6 to 21.4), required more surgeries (2.7 ± 1.3 versus 1.3 ± 0.8, p < .00001, CI 2.1 to 2.5), were 14 times more likely to have amputations (48.2% versus 6.5%, p < .0001, CI 3.0 to 60.2), and had 2 times longer hospital stays (16.2 ± 10.6 versus 7.5 ± 9 days, p = .0001, CI 11.9 to 15.9. Infected puncture wounds in patients with diabetes often fair much worse with more detrimental outcomes than those in patients without diabetes.

Erbium: Yttrium Aluminum Garnet Laser Accelerates Healing in Indolent Diabetic Foot Ulcers.

The objective of the study was to evaluate the effect of the erbium:yttrium aluminum garnet (YAG) laser on diabetic foot ulcers (DFUs) that had not responded to standard care. We retrospectively evaluated 22 nonhealing DFUs that received at least 4 weeks of standard wound care, demonstrated poor healing response, and subsequently were treated with an erbium:YAG laser. We measured the percent wound area reduction (PWAR) for the 4 weeks before initiating laser therapy and the PWAR for 4 weeks after the initiation of laser therapy. Erbium:YAG laser treatment consisted of 2 components: debridement and resurfacing. The laser settings were the same for all treatments. We used the paired t test to compare pretreatment with posttreatment wound area reduction. During the 4-week period before the initiation of laser therapy, the average PWAR was -33.6%. Four weeks after initiating treatment with the erbium:YAG laser, the average PWAR was 63.4% (p = .002) and 72.7% of wounds had ≥50% PWAR. By 12 weeks, 50% of wounds had healed. Erbium:YAG laser therapy accelerated DFU healing in a cohort of patients with ulcers that had been unresponsive to standard of care therapy.

Outcomes of Limb-Sparing Surgery for Osteomyelitis in the Diabetic Foot: Importance of the Histopathologic Margin.

BACKGROUND: Diabetes mellitus affects up to 14% of Americans. Infection of the diabetic foot is a common complication, which may lead to amputation. If infection extends to involve bone, the risk of amputation is increased 4-fold. Presence of osteomyelitis at the histopathologic margin of resection portends a poor prognosis in osteomyelitis outside the setting of the diabetic foot. We aimed to assess the association of a positive histopathologic margin with the outcome of osteomyelitis in the diabetic foot. METHODS: Medical records were reviewed for all patients who underwent below-ankle amputation for osteomyelitis of the diabetic foot. Patients who had at least 1 year of follow-up, a histopathologic diagnosis of osteomyelitis, and a comment on whether the margin was involved were included. RESULTS: Thirty-nine of 66 (59%) cases had remission of osteomyelitis at 12 months. When comparing cases with remission with those who experienced recurrence in the 12 months of follow-up, there were no statistically significant differences in age, glycosylated hemoglobin, duration of antimicrobial therapy, Infectious Diseases Society of America class, or presence of osteomyelitis at the histopathologic margin. Among cases with a negative histopathologic margin, 29/48 (60.4%) were free of disease at 1 year, compared with 10/18 (55.6%) cases with a positive histopathologic margin (P = .72). Remission was significantly more frequent in cases undergoing amputation at the digit level (66.7%) compared with amputation at the metatarsal level (40.7%) (P = .045). CONCLUSIONS: Osteomyelitis of the diabetic foot at the histopathologic margin of resection was not associated with increased risk of treatment failure. Resection at the level of the digit was associated with a lower risk of failure than at the metatarsal level.

Systems analysis of protective immune responses to RTS,S malaria vaccination in humans.

RTS,S is an advanced malaria vaccine candidate and confers significant protection against Plasmodium falciparum infection in humans. Little is known about the molecular mechanisms driving vaccine immunity. Here, we applied a systems biology approach to study immune responses in subjects receiving three consecutive immunizations with RTS,S (RRR), or in those receiving two immunizations of RTS,S/AS01 following a primary immunization with adenovirus 35 (Ad35) (ARR) vector expressing circumsporozoite protein. Subsequent controlled human malaria challenge (CHMI) of the vaccinees with Plasmodium-infected mosquitoes, 3 wk after the final immunization, resulted in ∼50% protection in both groups of vaccinees. Circumsporozoite protein (CSP)-specific antibody titers, prechallenge, were associated with protection in the RRR group. In contrast, ARR-induced lower antibody responses, and protection was associated with polyfunctional CD4+ T-cell responses 2 wk after priming with Ad35. Molecular signatures of B and plasma cells detected in PBMCs were highly correlated with antibody titers prechallenge and protection in the RRR cohort. In contrast, early signatures of innate immunity and dendritic cell activation were highly associated with protection in the ARR cohort. For both vaccine regimens, natural killer (NK) cell signatures negatively correlated with and predicted protection. These results suggest that protective immunity against P. falciparum can be achieved via multiple mechanisms and highlight the utility of systems approaches in defining molecular correlates of protection to vaccination.

Mental health symptom severity in cannabis using and non-using Veterans with probable PTSD.

BACKGROUND: Posttraumatic Stress Disorder (PTSD) is a disabling illness suffered by many Veterans returning from war. Some Veterans believe that cannabis may be therapeutic for PTSD. The purpose of this study was to better understand the association between cannabis use and PTSD symptoms. METHODS: The study was a matched case-control cross-sectional evaluation of the psychiatric and sociocultural associations of cannabis use in Veterans with probable PTSD. Patient self-report measures were examined comparing cannabis users (cases) to non-users (controls) who were case-matched on age and gender. RESULTS: Results indicated that there were no significant differences between cases and controls in mean PTSD Checklist-Civilian version (PCL-C) scores (59.2 and 59.1, respectively). There was also no association between PTSD scores and frequency of cannabis use. It was also observed that cases were more likely to be non-Caucasian, financially challenged, and unmarried. LIMITATIONS: The sample is a convenience sample of Veterans being referred for a clinical assessment and therefore, sampling biases may limit the generalizability of the results to other populations including Veterans not seeking health care in the Veterans Affairs (VA) system. CONCLUSIONS: The results do not support the theory that cannabis use would be associated with less severe PTSD symptoms. Results do suggest important sociocultural differences in cannabis users compared to controls.

Type I interferon-dependent activation of NK cells by rAd28 or rAd35, but not rAd5, leads to loss of vector-insert expression.

Vaccines constructed from rare-serotype recombinant adenovirus vectors (rAd) such as rAd serotype 28 (rAd28) and rAd35 are currently being explored as alternatives to rAd5-based vaccines because they circumvent the problems with pre-existing immunity that complicate the effectiveness of rAd5 vaccines. However, previous work has demonstrated that the immunogenicity of rAd28 and rAd35 is substantially lower than rAd5. Here we show that rAd28 and rAd35 increase apoptosis of antigen presenting cells (APCs), such as monocytes, relative to rAd5 and mock infected controls. APCs undergoing apoptosis showed an increased loss of vector-insert expression. Loss of vector-insert expression correlated with activation of NK cells, which resulted in apoptosis of co-cultured monocytes. Finally, we show that activation of NK cells is dependent on IFNα which is produced by exposure to rAd28 or rAd35, but not to rAd5. Taken together, these data demonstrate that IFNα-induced activation of NK cells leads to increased monocyte apoptosis and subsequent vector-insert loss. This may be a possible mechanism that results in reduced immunogenicity of rAd28 and rAd35-based vectors.

Type I IFN induced by adenovirus serotypes 28 and 35 has multiple effects on T cell immunogenicity.

Recombinant adenovirus (rAd) vectors are being investigated as vaccine delivery vehicles in preclinical and clinical studies. rAds constructed from different serotypes differ in receptor usage, tropism, and ability to activate cells, aspects of which likely contribute to their different immunogenicity profiles. In this study, we compared the infectivity and cell stimulatory capacity of recombinant adenovirus serotype 5 (rAd5), recombinant adenovirus serotype 28 (rAd28), and recombinant adenovirus serotype 35 (rAd35) in association with their respective immunogenicity profiles. We found that rAd28 and rAd35 infected and led to the in vitro maturation and activation of both human and mouse dendritic cells more efficiently compared with rAd5. In stark contrast to rAd5, rAd28 and rAd35 induced production of IFN-α and stimulated IFN-related intracellular pathways. However, the in vivo immunogenicity of rAd28 and rAd35 was significantly lower than that of rAd5. Deletion of IFN-α signaling during vaccination with rAd28 and rAd35 vectors increased the magnitude of the insert-specific T cell response to levels induced by vaccination with rAd5 vector. The negative impact of IFN-α signaling on the magnitude of the T cell response could be overcome by increasing the vaccine dose, which was also associated with greater polyfunctionality and a more favorable long-term memory phenotype of the CD8 T cell response in the presence of IFN-α signaling. Taken together, our results demonstrate that rAd-induced IFN-α production has multiple effects on T cell immunogenicity, the understanding of which should be considered in the design of rAd vaccine vectors.