Long-primed germinal centres with enduring affinity maturation and clonal migration.

Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.

Delayed vaginal SHIV infection in VRC01 and anti-α4ß7 treated rhesus macaques.

VRC01 protects macaques from vaginal SHIV infection after a single high-dose challenge. Infusion of a simianized anti-α4ß7 mAb (Rh-α4ß7) just prior to, and during repeated vaginal exposures to SIVmac251 partially protected macaques from vaginal SIV infection and rescued CD4+ T cells. To investigate the impact of combining VRC01 and Rh-α4ß7 on SHIV infection, 3 groups of macaques were treated with a suboptimal dosing of VRC01 alone or in combination with Rh-α4ß7 or with control antibodies prior to the initiation of weekly vaginal exposures to a high dose (1000 TCID50) of SHIVAD8-EO. The combination Rh-α4ß7-VRC01 significantly delayed SHIVAD8-EO vaginal infection. Following infection, VRC01-Rh-α4ß7-treated macaques maintained higher CD4+ T cell counts and exhibited lower rectal SIV-DNA loads compared to controls. Interestingly, VRC01-Rh-α4ß7-treated macaques had fewer IL-17-producing cells in the blood and the gut during the acute phase of infection. Moreover, higher T cell responses to the V2-loop of the SHIVAD8-EO envelope in the VRC01-Rh-α4ß7 group inversely correlated with set point viremia. The combination of suboptimal amounts of VRC01 and Rh-α4ß7 delayed infection, altered antiviral immune responses and minimized CD4+ T cell loss. Further exploration of the effect of combining bNAbs with Rh-α4ß7 on SIV/HIV infection and antiviral immune responses is warranted and may lead to novel preventive and therapeutic strategies.

Once-Weekly Oral Dosing of MK-8591 Protects Male Rhesus Macaques From Intrarectal Challenge With SHIV109CP3.

BACKGROUND: MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine [EFdA]) is a novel reverse transcriptase-translocation inhibitor. METHODS: We assessed MK-8591 as preexposure prophylaxis in the rhesus macaque model of intrarectal challenge with simian/human immunodeficiency virus (SHIV). In study 1, 8 rhesus macaques received 3.9 mg/kg of MK-8591 orally on day 0 and once weekly for the next 14 weeks. Eight controls were treated with vehicle. All rhesus macaques were challenged with SHIV109CP3 on day 6 and weekly for up to 12 challenges or until infection was confirmed. The dose of MK-8591 was reduced to 1.3 and 0.43 mg/kg/week in study 2 and further to 0.1 and 0.025 mg/kg/week in study 3. In studies 2 and 3, each dose was given up to 6 times once weekly, and animals were challenged 4 times once weekly with SHIV109CP3. RESULTS: Control macaques were infected after a median of 1 challenge (range, 1-4 challenges). All treated animals in studies 1 and 2 were protected, consistent with a 41.5-fold lower risk of infection (P < .0001, by the log-rank test). In study 3, at a 0.1-mg/kg dose, 2 rhesus macaques became infected, consistent with a 7.2-fold lower risk of infection (P = .0003, by the log-rank test). The 0.025-mg/kg dose offered no protection. CONCLUSIONS: These data support MK-8591's potential as a preexposure prophylaxis agent.

A Subcutaneous Implant of Tenofovir Alafenamide Fumarate Causes Local Inflammation and Tissue Necrosis in Rabbits and Macaques.

We describe the in vitro and in vivo evaluation of a subcutaneous reservoir implant delivering tenofovir alafenamide hemifumarate (TAF) for the prevention of HIV infection. These long-acting reservoir implants were able to deliver antiretroviral drug for over 90 days in vitro and in vivo We evaluated the implants for implantation site histopathology and pharmacokinetics in plasma and tissues for up to 12 weeks in New Zealand White rabbit and rhesus macaque models. A dose-ranging study in rabbits demonstrated dose-dependent pharmacokinetics and local inflammation up to severe necrosis around the active implants. The matched placebos showed normal wound healing and fibrous tissue encapsulation of the implant. We designed a second implant with a lower release rate and flux of TAF and achieved a median cellular level of tenofovir diphosphate of 42 fmol per 106 rhesus macaque peripheral blood mononuclear cells at a TAF dose of 10 µg/kg/day. This dose and flux of TAF also resulted in adverse local inflammation and necrosis near the implant in rhesus macaques. The level of inflammation in the primates was markedly lower in the placebo group than in the active-implant group. The histological inflammatory response to the TAF implant at 4 and 12 weeks in primates was graded as a severe reaction. Thus, while we were able to achieve a sustained target dose, we observed an unacceptable inflammatory response locally at the implant tissue interface.

Integrin α4ß7 Blockade Preferentially Impacts CCR6+ Lymphocyte Subsets in Blood and Mucosal Tissues of Naive Rhesus Macaques.

Infusion of a simianized anti-α4ß7 mAb (Rh-α4ß7) just before and following SIV infection protected rhesus macaques from developing AIDS and partially from vaginal SIV acquisition. Recently, short-term treatment with Rh-α4ß7 in combination with cART was found to lead to prolonged viral suppression after withdrawal of all therapeutic interventions. The humanized form of Rh-α4ß7, vedolizumab, is a highly effective treatment for inflammatory bowel disease. To clarify the mechanism of action of Rh-α4ß7, naive macaques were infused with Rh-α4ß7 and sampled in blood and tissues before and after treatment to monitor several immune cell subsets. In blood, Rh-α4ß7 increased the CD4+ and CD8+ T cell counts, but not B cell counts, and preferentially increased CCR6+ subsets while decreasing CD103+ and CD69+ lymphocytes. In mucosal tissues, surprisingly, Rh-α4ß7 did not impact integrin α4+ cells, but decreased the frequencies of CCR6+ and CD69+ CD4+ T cells and, in the gut, Rh-α4ß7 transiently decreased the frequency of memory and IgA+ B cells. In summary, even in the absence of inflammation, Rh-α4ß7 impacted selected immune cell subsets in different tissues. These data provide new insights into the mechanisms by which Rh-α4ß7 may mediate its effect in SIV-infected macaques with implications for understanding the effect of treatment with vedolizumab in patients with inflammatory bowel disease.

Correction: A Small Molecule, Which Competes with MAdCAM-1, Activates Integrin α4ß7 and Fails to Prevent Mucosal Transmission of SHIV-SF162P3.

[This corrects the article DOI: 10.1371/journal.ppat.1005720.].

A Small Molecule, Which Competes with MAdCAM-1, Activates Integrin α4ß7 and Fails to Prevent Mucosal Transmission of SHIV-SF162P3.

Mucosal HIV-1 transmission is inefficient. However, certain viral and host characteristics may play a role in facilitating HIV acquisition and systemic expansion. Cells expressing high levels of integrin α4ß7 have been implicated in favoring the transmission process and the infusion of an anti-α4ß7 mAb (RM-Act-1) prior to, and during a repeated low-dose vaginal challenge (RLDC) regimen with SIVmac251 reduced SIV acquisition and protected the gut-associated lymphoid tissues (GALT) in the macaques that acquired SIV. α4ß7 expression is required for lymphocyte trafficking to the gut lamina propria and gut inductive sites. Several therapeutic strategies that target α4ß7 have been shown to be effective in treating inflammatory conditions of the intestine, such as inflammatory bowel disease (IBD). To determine if blocking α4ß7 with ELN, an orally available anti-α4 small molecule, would inhibit SHIV-SF162P3 acquisition, we tested its ability to block MAdCAM-1 (α4ß7 natural ligand) and HIV-gp120 binding in vitro. We studied the pharmacokinetic profile of ELN after oral and vaginal delivery in macaques. Twenty-six macaques were divided into 3 groups: 9 animals were treated with ELN orally, 9 orally and vaginally and 8 were used as controls. All animals were challenged intra-vaginally with SHIV-SF162P3 using the RLDC regimen. We found that ELN did not protect macaques from SHIV acquisition although it reduced the SHIV-induced inflammatory status during the acute phase of infection. Notably, integrins can exist in different activation states and, comparing the effect of ELN and the anti-α4ß7 mAb RM-Act-1 that reduced susceptibility to SIV infection, we determined that ELN induces the active conformation of α4ß7, while RM-Act-1 inhibits its activation through an allosteric mechanism. These results suggest that inhibition of α4ß7 activation may be necessary to reduce susceptibility to SIV/SHIV infection and highlight the complexity of anti-integrins therapeutic approach in HIV as well as in IBD and other autoimmune diseases.

A model of genital herpes simplex virus Type 1 infection in Rhesus Macaques.

BACKGROUND: Although HSV-2 is the major cause of genital lesions, HSV-1 accounts for half of new cases in developed countries. METHODS: Three healthy SHIV-SF162P3-infected Indian rhesus macaques were inoculated with 4×108 pfu of HSV-1 twice, with the second inoculation performed after the vaginal mucosa was gently abraded with a cytobrush. RESULTS: HSV-1 DNA was detected in vaginal swabs 5 days after the second but not the first inoculation in all three macaques. An increase in inflammatory cytokines was detected in the vaginal fluids of the animals with no or intermittent shedding. Higher frequency of blood α4 ß7high CD4+ T cells was measured in the animals with consistent and intermitted shedding, while a decrease in the frequency of CD69+ CD4+ T cells was present in all animals. CONCLUSIONS: This macaque model of genital HSV-1 could be useful to study the impact of the growing epidemic of genital HSV-1 on HIV infection.

High IL-1ß and IL-18 Levels Associate with Gut Barrier Disruption and Monocyte Activation During Chronic SIV Infection with Long-Term Suppressive Antiretroviral Therapy.

HIV-induced persistent immune activation is a key mediator of inflammatory comorbidities such as cardiovascular disease (CVD) and neurocognitive disorders. While a preponderance of data indicate that gut barrier disruption and microbial translocation are drivers of chronic immune activation, the molecular mechanisms of this persistent inflammatory state remain poorly understood. Here, utilizing the nonhuman primate model of HIV infection with suppressive antiretroviral therapy (ART), we investigated activation of inflammasome pathways and their association with intestinal epithelial barrier disruption and CVD pathogenesis. Longitudinal blood samples obtained from rhesus macaques with chronic SIV infection and long-term suppressive ART were evaluated for biomarkers of intestinal epithelial barrier disruption (IEBD), inflammasome activation (IL-1ß and IL-18), inflammatory cytokines, and triglyceride (TG) levels. Activated monocyte subpopulations and glycolytic potential were investigated in peripheral blood mononuclear cells (PBMCs). Higher plasma levels of IL-1ß and IL-18 were observed following the hallmark increase in IEBD biomarkers, intestinal fatty acid-binding protein (IFABP) and LPS-binding protein (LBP), during the chronic phase of treated SIV infection. Further, significant correlations of plasma IFABP levels with IL-1ß and IL-18 were observed between 10-12 months of ART. Higher levels of sCD14, IL-6, and GM-CSF, among other inflammatory mediators, were also observed only during the long-term SIV+ART phase along with a trend of increase in frequencies of activated CD14 + CD16 + intermediate monocyte subpopulations. Lastly, we found elevated levels of blood TG and higher glycolytic capacity in PBMCs of chronic SIV-infected macaques with long-term ART. The increase in circulating IL-18 and IL-1ß following IEBD and their significant positive correlation with IFABP suggest a connection between gut barrier disruption and inflammasome activation during chronic SIV infection, despite viral suppression with ART. Additionally, the increase in markers of monocyte activation, along with elevated TG and enhanced glycolytic pathway activity, indicates metabolic remodeling that could accelerate CVD pathogenesis. Further research is needed to understand mechanisms by which gut dysfunction and inflammasome activation contribute to HIV-associated CVD and metabolic complications, enabling targeted interventions in people with HIV.

A combined adjuvant approach primes robust germinal center responses and humoral immunity in non-human primates.

Adjuvants and antigen delivery kinetics can profoundly influence B cell responses and should be critically considered in rational vaccine design, particularly for difficult neutralizing antibody targets such as human immunodeficiency virus (HIV). Antigen kinetics can change depending on the delivery method. To promote extended immunogen bioavailability and to present antigen in a multivalent form, native-HIV Env trimers are modified with short phosphoserine peptide linkers that promote tight binding to aluminum hydroxide (pSer:alum). Here we explore the use of a combined adjuvant approach that incorporates pSer:alum-mediated antigen delivery with potent adjuvants (SMNP, 3M-052) in an extensive head-to-head comparison study with conventional alum to assess germinal center (GC) and humoral immune responses. Priming with pSer:alum plus SMNP induces additive effects that enhance the magnitude and persistence of GCs, which correlate with better GC-TFH cell help. Autologous HIV-neutralizing antibody titers are improved in SMNP-immunized animals after two immunizations. Over 9 months after priming immunization of pSer:alum with either SMNP or 3M-052, robust Env-specific bone marrow plasma cells (BM BPC) are observed. Furthermore, pSer-modification of Env trimer reduce targeting towards immunodominant non-neutralizing epitopes. The study shows that a combined adjuvant approach can augment humoral immunity by modulating immunodominance and shows promise for clinical translation.

Impact of SIV infection on mycobacterial lipid-reactive T cell responses in Bacillus Calmette-Guérin (BCG) inoculated macaques.

Background: Although BCG vaccine protects infants from tuberculosis (TB), it has limited efficacy in adults against pulmonary TB. Further, HIV coinfection significantly increases the risk of developing active TB. In the lack of defined correlates of protection in TB disease, it is essential to explore immune responses beyond conventional CD4 T cells to gain a better understanding of the mechanisms of TB immunity. Methods: Here, we evaluated unconventional lipid-reactive T cell responses in cynomolgus macaques following aerosol BCG inoculation and examined the impact of subsequent SIV infection on these responses. Immune responses to cellular lipids of M. bovis and M. tuberculosis were examined ex vivo in peripheral blood and bronchioalveolar lavage (BAL). Results: Prior to BCG inoculation, innate-like IFN-γ responses to mycobacterial lipids were observed in T cells. Aerosol BCG exposure induced an early increase in frequencies of BAL γδT cells, a dominant subset of lipid-reactive T cells, along with enhanced IL-7R and CXCR3 expression. Further, BCG exposure stimulated greater IFN-γ responses to mycobacterial lipids in peripheral blood and BAL, suggesting the induction of systemic and local Th1-type response in lipid-reactive T cells. Subsequent SIV infection resulted in a significant loss of IL-7R expression on blood and BAL γδT cells. Additionally, IFN-γ responses of mycobacterial lipid-reactive T cells in BAL fluid were significantly lower in SIV-infected macaques, while perforin production was maintained through chronic SIV infection. Conclusions: Overall, these data suggest that despite SIV-induced decline in IL-7R expression and IFN-γ production by mycobacterial lipid-reactive T cells, their cytolytic potential is maintained. A deeper understanding of anti-mycobacterial lipid-reactive T cell functions may inform novel approaches to enhance TB control in individuals with or without HIV infection.

Recombinant Simian Varicella Virus-Simian Immunodeficiency Virus Vaccine Induces T and B Cell Functions and Provides Partial Protection against Repeated Mucosal SIV Challenges in Rhesus Macaques.

HIV vaccine mediated efficacy, using an expanded live attenuated recombinant varicella virus-vectored SIV rSVV-SIVgag/env vaccine prime with adjuvanted SIV-Env and SIV-Gag protein boosts, was evaluated in a female rhesus macaques (RM) model against repeated intravaginal SIV challenges. Vaccination induced anti-SIV IgG responses and neutralizing antibodies were found in all vaccinated RMs. Three of the eight vaccinated RM remained uninfected (vaccinated and protected, VP) after 13 repeated challenges with the pathogenic SIVmac251-CX-1. The remaining five vaccinated and infected (VI) macaques had significantly reduced plasma viral loads compared with the infected controls (IC). A significant increase in systemic central memory CD4+ T cells and mucosal CD8+ effector memory T-cell responses was detected in vaccinated RMs compared to controls. Variability in lymph node SIV-Gag and Env specific CD4+ and CD8+ T cell cytokine responses were detected in the VI RMs while all three VP RMs had more durable cytokine responses following vaccination and prior to challenge. VI RMs demonstrated predominately SIV-specific monofunctional cytokine responses while the VP RMs generated polyfunctional cytokine responses. This study demonstrates that varicella virus-vectored SIV vaccination with protein boosts induces a 37.5% efficacy rate against pathogenic SIV challenge by generating mucosal memory, virus specific neutralizing antibodies, binding antibodies, and polyfunctional T-cell responses.

Viability of acellular biologic graft for nipple-areolar complex reconstruction in a non-human primate model.

Many of the > 3.5 million breast cancer survivors in the US have undergone breast reconstruction following mastectomy. Patients report that nipple-areolar complex (NAC) reconstruction is psychologically important, yet current reconstruction techniques commonly result in inadequate shape, symmetry, and nipple projection. Our team has developed an allogeneic acellular graft for NAC reconstruction (dcl-NAC) designed to be easy to engraft, lasting, and aesthetically pleasing. Here, dcl-NAC safety and host-mediated re-cellularization was assessed in a 6-week study in rhesus macaque non-human primates (NHPs). Human-derived dcl-NACs (n = 30) were engrafted on the dorsum of two adult male NHPs with each animal's own nipples as controls (n = 4). Weight, complete blood counts, and metabolites were collected weekly. Grafts were removed at weeks 1, 3, or 6 post-engraftment for histology. The primary analysis evaluated health, re-epithelialization, and re-vascularization. Secondary analysis evaluated re-innervation. Weight, complete blood counts, and metabolites remained mostly within normal ranges. A new epidermal layer was observed to completely cover the dcl-NAC surface at week 6 (13-100% coverage, median 93.3%) with new vasculature comparable to controls at week 3 (p = 0.10). Nerves were identified in 75% of dcl-NACs (n = 9/12) at week 6. These data suggest that dcl-NAC is safe and supports host-mediated re-cellularization.

Gut Microbiome Changes Associated with Epithelial Barrier Damage and Systemic Inflammation during Antiretroviral Therapy of Chronic SIV Infection.

Gut dysbiosis is a common feature associated with the chronic inflammation of HIV infection. Toward understanding the interplay of chronic treated HIV infection, dysbiosis, and systemic inflammation, we investigated longitudinal fecal microbiome changes and plasma inflammatory markers in the nonhuman primate model. Following simian immunodeficiency virus (SIV) infection in rhesus macaques, significant changes were observed in several members of the phylum Firmicutes along with an increase in Bacteroidetes. Viral suppression with antiretroviral therapy (ART) resulted in an early but partial recovery of compositional changes and butyrate producing genes in the gut microbiome. Over the course of chronic SIV infection and long-term ART, however, the specific loss of Faecalibacterium prausnitzii and Treponema succinifaciens significantly correlated with an increase in plasma inflammatory cytokines including IL-6, G-CSF, I-TAC, and MIG. Further, the loss of T. succinifaciens correlated with an increase in circulating biomarkers of gut epithelial barrier damage (IFABP) and microbial translocation (LBP and sCD14). As F. prausnitzii and T. succinifaciens are major short-chain fatty acid producing bacteria, their sustained loss during chronic SV-ART may contribute to gut inflammation and metabolic alterations despite effective long-term control of viremia. A better understanding of the correlations between the anti-inflammatory bacterial community and healthy gut barrier functions in the setting of long-term ART may have a major impact on the clinical management of inflammatory comorbidities in HIV-infected individuals.

Dysregulation of IL-17/IL-22 Effector Functions in Blood and Gut Mucosal Gamma Delta T Cells Correlates With Increase in Circulating Leaky Gut and Inflammatory Markers During cART-Treated Chronic SIV Infection in Macaques.

HIV-associated inflammation has been implicated in the premature aging and increased risk of age-associated comorbidities in cART-treated individuals. However, the immune mechanisms underlying the chronic inflammatory state of cART-suppressed HIV infection remain unclear. Here, we investigated the role of γδT cells, a group of innate IL-17 producing T lymphocytes, in the development of systemic inflammation and leaky gut phenotype during cART-suppressed SIV infection of macaques. Plasma levels of inflammatory mediators, intestinal epithelial barrier disruption (IEBD) and microbial translocation (MT) biomarkers, and Th1/Th17-type cytokine functions were longitudinally assessed in blood and gut mucosa of SIV-infected, cART-suppressed macaques. Among the various gut mucosal IL-17/IL-22-producing T lymphocyte subsets including Th17, γδT, CD161+ CD8+ T, and MAIT cells, a specific decline in the Vδ2 subset of γδT cells and impaired IL-17/IL-22 production in γδT cells significantly correlated with the subsequent increase in plasma IEBD/MT markers (IFABP, LPS-binding protein, and sCD14) and pro-inflammatory cytokines (IL-6, IL-1ß, IP10, etc.) despite continued viral suppression during long-term cART. Further, the plasma inflammatory cytokine signature during long-term cART was distinct from acute SIV infection and resembled the inflammatory cytokine profile of uninfected aging (inflammaging) macaques. Overall, our data suggest that during cART-suppressed chronic SIV infection, dysregulation of IL-17/IL-22 cytokine effector functions and decline of Vδ2 γδT cell subsets may contribute to gut epithelial barrier disruption and development of a distinct plasma inflammatory signature characteristic of inflammaging. Our results advance the current understanding of the impact of chronic HIV/SIV infection on γδT cell functions and demonstrate that in the setting of long-term cART, the loss of epithelial barrier-protective functions of Vδ2 T cells and ensuing IEBD/MT occurs before the hallmark expansion of Vδ1 subsets and skewed Vδ2/Vδ1 ratio. Thus, our work suggests that novel therapeutic approaches toward restoring IL-17/IL-22 cytokine functions of intestinal Vδ2 T cells may be beneficial in preserving gut epithelial barrier function and reducing chronic inflammation in HIV-infected individuals.

Blocking α4ß7 integrin delays viral rebound in SHIVSF162P3-infected macaques treated with anti-HIV broadly neutralizing antibodies.

Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α4ß7 with an anti-α4ß7 monoclonal antibody (Rh-α4ß7) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α4ß7 integrin blockade, SHIVSF162P3-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α4ß7 or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-α4ß7 and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that α4ß7 integrin blockade may prolong virologic control by bNAbs in SHIVSF162P3-infected macaques.

Anti-α4ß7 monoclonal antibody-conjugated nanoparticles block integrin α4ß7 on intravaginal T cells in rhesus macaques.

Intravenous administration of anti-α4ß7 monoclonal antibody in macaques decreases simian immunodeficiency virus (SIV) vaginal infection and reduces gut SIV loads. Because of potential side effects of systemic administration, a prophylactic strategy based on mucosal administration of anti-α4ß7 antibody may be safer and more effective. With this in mind, we developed a novel intravaginal formulation consisting of anti-α4ß7 monoclonal antibody-conjugated nanoparticles (NPs) loaded in a 1% hydroxyethylcellulose (HEC) gel (NP-α4ß7 gel). When intravaginally administered as a single dose in a rhesus macaque model, the formulation preferentially bound to CD4+ or CD3+ T cells expressing high levels of α4ß7, and occupied ~40% of α4ß7 expressed by these subsets and ~25% of all cells expressing α4ß7 Blocking of the α4ß7 was restricted to the vaginal tract without any changes detected systemically.

Acellular Biologic Nipple-Areolar Complex Graft: In Vivo Murine and Nonhuman Primate Host Response Evaluation.

There are more than 3 million breast cancer survivors living in the United States of which a significant number have undergone mastectomy followed by breast and nipple-areolar complex (NAC) reconstruction. Current strategies for NAC reconstruction are dependent on nonliving or nonpermanent techniques, including tattooing, nipple prosthetics, or surgical nipple-like structures. Described herein is a tissue engineering approach demonstrating the feasibility of an allogeneic acellular graft for nipple reconstruction. Nonhuman primate (NHP)-derived NAC tissues were decellularized and their extracellular matrix components analyzed by both proteomic and histological analyses. Decellularized NHP nipple tissue showed the removal of intact cells and greatly diminished profiles for intracellular proteins, as compared with intact NHP nipple tissue. We further evaluated the biocompatibility of decellularized grafts and their potential to support host-mediated neovascularization against commercially available acellular dermal grafts by performing in vivo studies in a murine model. A follow-up NHP pilot study evaluated the host-mediated neovascularization and re-epithelialization of onlay engrafted decellularized NAC grafts. The murine model revealed greater neovascularization in the decellularized NAC than in the commercially available control grafts, with no observed biocompatibility issues. The in vivo NHP model confirmed that the decellularized NAC grafts encourage neovascularization as well as re-epithelialization. These results support the concept that a biologically derived acellular nipple graft is a feasible approach for nipple reconstruction, supporting neovascularization in the absence of adverse systemic responses. Impact statement Currently, women in the United States most often undergo a mastectomy, followed by reconstruction, after being diagnosed with breast cancer. These breast cancer survivors are often left with nipple-areolar complex (NAC) reconstructions that are subsatisfactory, nonliving, and/or nonpermanent. Utilizing an acellular biologically derived whole NAC graft would allow these patients a living and permanent tissue engineering solution to nipple reconstruction.

Evaluation of the host immune response to decellularized lung scaffolds derived from α-Gal knockout pigs in a non-human primate model.

Whole organ tissue engineering is a promising approach to address organ shortages in many applications, including lung transplantation for patients with chronic pulmonary disease. Engineered lungs may be derived from animal sources after removing cellular content, exposing the extracellular matrix to serve as a scaffold for recellularization with human cells. However, the use of xenogeneic tissue sources in human transplantation raises concerns due to the presence of the antigenic Gal epitope. In the present study, lungs from wild type or α-Gal knockout pigs were harvested, decellularized, and implanted subcutaneously in a non-human primate model to evaluate the host immune response. The decellularized porcine implants were compared to a sham surgery control, as well as native porcine and decellularized macaque lung implants. The results demonstrated differential profiles of circulating and infiltrating immune cell subsets and histological outcomes depending on the implanted tissue source. Upon implantation, the decellularized α-Gal knockout lung constructs performed similarly to the decellularized wild type lung constructs. However, upon re-implantation into a chronic exposure model, the decellularized wild type lung constructs resulted in a greater proportion of infiltrating CD45+ cells, including CD3+ and CD8+ cytotoxic T-cells, likely mediated by an increase in production of Gal-specific antibodies. The results suggest that removal of the Gal epitope can potentially reduce adverse inflammatory reactions associated with chronic exposure to engineered organs containing xenogeneic components.