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Sterile inflammation is the immune response to damage-associated molecular patterns (DAMPs) released during cell death in the absence of foreign pathogens. In the setting of solid organ transplantation, ischemia-reperfusion injury results in mitochondria-mediated production of reactive oxygen and nitrogen species that are a major cause of uncontrolled cell death and release of various DAMPs from the graft tissue. When properly regulated, the immune response initiated by DAMP-sensing serves as means of damage control and is necessary for initiation of recovery pathways and re-establishment of homeostasis. In contrast, a dysregulated or overt sterile inflammatory response can inadvertently lead to further injury through recruitment of immune cells, innate immune cell activation, and sensitization of the adaptive immune system. In liver transplantation, sterile inflammation may manifest as early graft dysfunction, acute graft failure, or increased risk of immunosuppression-resistant rejection. Understanding the mechanisms of the development of sterile inflammation in the setting of liver transplantation is crucial for finding reliable biomarkers that predict graft function, and for development of therapeutic approaches to improve long-term transplant outcomes. Here, we discuss the recent advances that have been made to elucidate the early signs of sterile inflammation and extent of damage from it. We also discuss new therapeutics that may be effective in quelling the detrimental effects of sterile inflammation.

Subnormothermic machine perfusion (SNMP) of liver grafts is currently less clinically developed than normothermic and hypothermic approaches, but may have logistical advantages. At intermediate temperatures, the oxygen demand of the graft is low enough to be satisfied with an acellular perfusate, obviating the need for oxygen carrying molecules. This intermediate metabolic rate, however, is sufficient to support the production of bile, which is emerging as an important indicator of graft injury and viability. In this study, we hypothesized that the biliary compartment would be more sensitive than perfusate in detecting graft injury during SNMP. To test this hypothesis in a rat model, we performed liver transplants with DCD and control liver grafts after 1 h of acellular room temperature machine perfusion (acRTMP) or static cold storage (SCS). Point of care liver function tests were measured in biliary and perfusate samples after 1 h of machine perfusion. Following transplantation, rats were sacrificed at 24 h for assessment of post-transplant graft function and histology. All point-of-care liver function tests were significantly more concentrated in the biliary compartment than the perfusate compartment during acRTMP. DCD liver grafts could be distinguished from control liver grafts by significantly higher markers of hepatocyte injury (AST, ALT) in the biliary compartment, but not in the perfusate compartment. Classical markers of cholangiocyte injury, such as gammy-glut amyl transferase (GGT), amylase (AML), and alkaline phosphatase were detectable in the biliary compartment, but not in the perfusate compartment. In comparison to SCS, graft preservation by acRTMP produced a significant survival benefit in DCD liver transplantation (75 vs. 0%, < 0.0030). Together, these findings demonstrate that during acRTMP, the biliary compartment may be a more sensitive indicator of graft injury than the perfusate compartment. Moreover, acRTMP provides superior graft preservation to SCS in rat DCD liver transplantation.

Without novel strategies to prevent mother-to-child HIV-1 transmission, more than 5% of HIV-1-exposed infants will continue to acquire HIV-1, most through breastfeeding. This study of rhesus macaque dam-and-infant pairs is the first preclinical study to investigate the protective role of transplacentally transferred HIV-1 vaccine-elicited antibodies and HIV-1 vaccine-elicited breast milk antibody responses in infant oral virus acquisition. It revealed highly variable placental transfer of potentially protective antibodies and emphasized the importance of pregnancy immunization timing to reach peak antibody levels prior to delivery. While there was no discernible impact of maternal immunization on late infant oral virus acquisition, we observed a strong correlation between the percentage of activated CD4 + T cells in infant peripheral blood and a reduced number of challenges to infection. This finding highlights an important consideration for future studies evaluating alternative strategies to further reduce the vertical HIV-1 transmission risk. Mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) contributes to an estimated 150,000 new infections annually. Maternal vaccination has proven safe and effective at mitigating the impact of other neonatal pathogens and is one avenue toward generating the potentially protective immune responses necessary to inhibit HIV-1 infection of infants through breastfeeding. In the present study, we tested the efficacy of a maternal vaccine regimen consisting of a modified vaccinia virus Ankara (MVA) 1086.C gp120 prime-combined intramuscular-intranasal gp120 boost administered during pregnancy and postpartum to confer passive protection on infant rhesus macaques against weekly oral exposure to subtype C simian-human immunodeficiency virus 1157ipd3N4 (SHIV1157ipd3N4) starting 6 weeks after birth. Despite eliciting a robust systemic envelope (Env)-specific IgG response, as well as durable milk IgA responses, the maternal vaccine did not have a discernible impact on infant oral SHIV acquisition. This study revealed considerable variation in vaccine-elicited IgG placental transfer and a swift decline of both Env-specific antibodies (Abs) and functional Ab responses in the infants prior to the first challenge, illustrating the importance of pregnancy immunization timing to elicit optimal systemic Ab levels at birth. Interestingly, the strongest correlation to the number of challenges required to infect the infants was the percentage of activated CD4 + T cells in the infant peripheral blood at the time of the first challenge. These findings suggest that, in addition to maternal immunization, interventions that limit the activation of target cells that contribute to susceptibility to oral HIV-1 acquisition independently of vaccination may be required to reduce infant HIV-1 acquisition via breastfeeding. IMPORTANCE Without novel strategies to prevent mother-to-child HIV-1 transmission, more than 5% of HIV-1-exposed infants will continue to acquire HIV-1, most through breastfeeding. This study of rhesus macaque dam-and-infant pairs is the first preclinical study to investigate the protective role of transplacentally transferred HIV-1 vaccine-elicited antibodies and HIV-1 vaccine-elicited breast milk antibody responses in infant oral virus acquisition. It revealed highly variable placental transfer of potentially protective antibodies and emphasized the importance of pregnancy immunization timing to reach peak antibody levels prior to delivery. While there was no discernible impact of maternal immunization on late infant oral virus acquisition, we observed a strong correlation between the percentage of activated CD4 + T cells in infant peripheral blood and a reduced number of challenges to infection. This finding highlights an important consideration for future studies evaluating alternative strategies to further reduce the vertical HIV-1 transmission risk.

The benefits of mucosal vaccines over injected vaccines are difficult to ascertain since mucosally administered vaccines often induce serum antibody responses of lower magnitude than those induced by injected vaccines. This study aimed to determine if mucosal vaccination using a modified vaccinia Ankara expressing HIV-1 gp120 (MVA-g120) prime and HIV-1 gp120 protein boost could be optimized to induce serum antibody responses similar to those induced by an intramuscularly (IM) administered MVA prime/gp120 boost to allow comparison of an IM immunization regimen to a mucosal vaccination regimen for their ability to protect against a low dose rectal SHIV challenge while inducing similar serum anti-HIV-1 antibody responses. A 3-fold higher antigen dose was required for intranasal (IN) immunization with gp120 to induce serum anti-gp120 IgG responses not significantly different than those induced by IM immunization. Gp120 fused to the Adenovirus type 2 fiber binding domain (gp120-Ad2F), a mucosal targeting ligand, exhibited enhanced IN immunogenicity when compared to gp120 alone. MVA-gp120 was more immunogenic after IN delivery than gastric or rectal delivery, although serum antibodies induced by IN immunization were lower than those induced by intramuscular immunization. Using these optimized vaccines, an IN MVA-gp120 prime, combined IM (gp120) and IN (gp120-Ad2F) boost regimen (IN/IM+IN) induced serum anti-gp120 antibody titers similar to those induced by the intramuscular prime/boost regimen (IM/IM) in rabbits and non-human primates. Despite the induction of similar systemic anti-HIV-1 antibody responses, neither the IM/IM nor the IN/IM+IN regimen induced elevated anti-HIV-1 mucosal IgA responses nor protected against a repeated low-dose rectal SHIV challenge. These results demonstrate that immunization regimens utilizing the IN route are able to induce serum antigen-specific antibody responses similar to those induced by systemic immunization.

The RV144 Thai trial HIV-1 vaccine of recombinant poxvirus (ALVAC) and recombinant HIV-1 gp120 subtype B/subtype E (B/E) proteins demonstrated 31% vaccine efficacy. Here we design an ALVAC/Pentavalent B/E/E/E/E vaccine to increase the diversity of gp120 motifs in the immunogen to elicit a broader antibody response and enhance protection. We find that immunization of rhesus macaques with the pentavalent vaccine results in protection of 55% of pentavalent-vaccine-immunized macaques from simian–human immunodeficiency virus (SHIV) challenge. Systems serology of the antibody responses identifies plasma antibody binding to HIV-infected cells, peak ADCC antibody titres, NK cell-mediated ADCC and antibody-mediated activation of MIP-1β in NK cells as the four immunological parameters that best predict decreased infection risk that are improved by the pentavalent vaccine. Thus inclusion of additional gp120 immunogens to a pox-prime/protein boost regimen can augment antibody responses and enhance protection from a SHIV challenge in rhesus macaques. A previous human HIV-1 vaccine clinical trial, boosting with HIV envelope protein from two strains, demonstrated moderate vaccine efficacy. Here, Bradley et al . show that a pentavalent HIV envelope protein boost improves protection from viral challenge in non-human primates and they identify immune correlates of protection.

Cytomegalovirus (CMV) is the most common congenital infection and cause of birth defects worldwide. Primary CMV infection during pregnancy leads to a higher frequency of congenital CMV (cCMV) than maternal re-infection, suggesting that maternal immunity confers partial protection. However, poorly understood immune correlates of protection against placental transmission contributes to the current lack of an approved vaccine to prevent cCMV. In this study, we characterized the kinetics of maternal plasma rhesus CMV (RhCMV) viral load (VL) and RhCMV-specific antibody binding and functional responses in a group of 12 immunocompetent dams with acute, primary RhCMV infection. We defined cCMV transmission as RhCMV detection in amniotic fluid (AF) by qPCR. We then leveraged a large group of past and current primary RhCMV infection studies in late-first/early-second trimester RhCMV-seronegative rhesus macaque dams, including immunocompetent (n = 15), CD4+ T cell-depleted with (n = 6) and without (n = 6) RhCMV-specific polyclonal IgG infusion before infection to evaluate differences between RhCMV AF-positive and AF-negative dams. During the first 3 weeks after infection, the magnitude of RhCMV VL in maternal plasma was higher in AF-positive dams in the combined cohort, while RhCMV glycoprotein B (gB)- and pentamer-specific binding IgG responses were lower magnitude compared to AF-negative dams. However, these observed differences were driven by the CD4+ T cell-depleted dams, as there were no differences in plasma VL or antibody responses between immunocompetent AF-positive vs AF-negative dams. Overall, these results suggest that levels of neither maternal plasma viremia nor humoral responses are associated with cCMV following primary maternal infection in healthy individuals. We speculate that other factors related to innate immunity are more important in this context as antibody responses to acute infection likely develop too late to influence vertical transmission. Yet, pre-existing CMV glycoprotein-specific and neutralizing IgG may provide protection against cCMV following primary maternal CMV infection even in high-risk, immunocompromised settings.

Human cytomegalovirus (HCMV) is the most common vertically transmitted infection worldwide, yet there are no vaccines or therapeutics to prevent congenital HCMV (cCMV) infection. Emerging evidence indicates that antibody Fc effector functions may be a previously underappreciated component of maternal immunity against HCMV. We recently reported that antibody-dependent cellular phagocytosis (ADCP) and IgG activation of FcγRI/FcγRII were associated with protection against cCMV transmission, leading us to hypothesize that additional Fc-mediated antibody functions may be important. In this same cohort of HCMV-transmitting (n = 41) and nontransmitting (n = 40) mother-infant dyads, we report that higher maternal sera antibody-dependent cellular cytotoxicity (ADCC) activation is also associated with lower risk of cCMV transmission. We investigated the relationship between ADCC and IgG responses against 9 viral antigens and found that ADCC activation correlated most strongly with sera IgG binding to the HCMV immunoevasin protein UL16. Moreover, we determined that higher UL16-specific IgG binding and FcγRIII/CD16 engagement were associated with the greatest risk reduction in cCMV transmission. Our findings indicate that ADCC-activating antibodies against targets such as UL16 may represent an important protective maternal immune response against cCMV infection that can guide future HCMV correlates studies and vaccine or antibody-based therapeutic development.

The canary pox vector and gp120 vaccine (ALVAC-HIV and AIDSVAX B/E gp120) in the RV144 HIV-1 vaccine trial conferred an estimated 31% vaccine efficacy. Although the vaccine Env AE.A244 gp120 is antigenic for the unmutated common ancestor of V1V2 broadly neutralizing antibody (bnAbs), no plasma bnAb activity was induced. The RV305 (NCT01435135) HIV-1 clinical trial was a placebo-controlled randomized double-blinded study that assessed the safety and efficacy of vaccine boosting on B cell repertoires. HIV-1-uninfected RV144 vaccine recipients were reimmunized 6-8 years later with AIDSVAX B/E gp120 alone, ALVAC-HIV alone, or a combination of ALVAC-HIV and AIDSVAX B/E gp120 in the RV305 trial. Env-specific post-RV144 and RV305 boost memory B cell VH mutation frequencies increased from 2.9% post-RV144 to 6.7% post-RV305. The vaccine was well tolerated with no adverse events reports. While post-boost plasma did not have bnAb activity, the vaccine boosts expanded a pool of envelope CD4 binding site (bs)-reactive memory B cells with long third heavy chain complementarity determining regions (HCDR3) whose germline precursors and affinity matured B cell clonal lineage members neutralized the HIV-1 CRF01 AE tier 2 (difficult to neutralize) primary isolate, CNE8. Electron microscopy of two of these antibodies bound with near-native gp140 trimers showed that they recognized an open conformation of the Env trimer. Although late boosting of RV144 vaccinees expanded a novel pool of neutralizing B cell clonal lineages, we hypothesize that boosts with stably closed trimers would be necessary to elicit antibodies with greater breadth of tier 2 HIV-1 strains. ClinicalTrials.gov NCT01435135.

Novel interventions that do not rely on daily adherence to ART are needed to achieve sustained viral remission for perinatally infected children, who currently rely on lifelong ART. Considering the risks and expense associated with ART interruption trials, the identification of biomarkers of viral rebound will prioritize promising therapeutic intervention strategies, including anti-HIV Env protein therapeutics. However, comprehensive studies to identify those biomarkers are logistically challenging in human infants, demanding the need for relevant nonhuman primate models of HIV rebound. In this study, we developed an infant RM model of oral infection with simian-human immunodeficiency virus expressing clade C HIV Env and short-term ART followed by ATI, longitudinally characterizing the immune responses to viral infection during ART and after ATI. Additionally, we compared this infant RM model to an analogous adult RM rebound model and identified virologic and immunologic correlates of the time to viral rebound after ATI. To achieve long-term viral remission in human immunodeficiency virus (HIV)-infected children, novel strategies beyond early antiretroviral therapy (ART) will be necessary. Identifying clinical predictors of the time to viral rebound upon ART interruption will streamline the development of novel therapeutic strategies and accelerate their evaluation in clinical trials. However, identification of these biomarkers is logistically challenging in infants, due to sampling limitations and the potential risks of treatment interruption. To facilitate the identification of biomarkers predicting viral rebound, we have developed an infant rhesus macaque (RM) model of oral simian-human immunodeficiency virus (SHIV) SHIV.CH505.375H.dCT challenge and analytical treatment interruption (ATI) after short-term ART. We used this model to characterize SHIV replication kinetics and virus-specific immune responses during short-term ART or after ATI and demonstrated plasma viral rebound in 5 out of 6 (83%) infants. We observed a decline in humoral immune responses and partial dampening of systemic immune activation upon initiation of ART in these infants. Furthermore, we monitored SHIV replication and rebound kinetics in infant and adult RMs and found that both infants and adults demonstrated equally potent virus-specific humoral immune responses. Finally, we validated our models by confirming a well-established correlate of the time to viral rebound, namely, the pre-ART plasma viral load, as well as identified additional potential humoral immune correlates. Thus, this model of infant ART and viral rebound can be used and further optimized to define biomarkers of viral rebound following long-term ART as well as to preclinically assess novel therapies to achieve a pediatric HIV functional cure. IMPORTANCE Novel interventions that do not rely on daily adherence to ART are needed to achieve sustained viral remission for perinatally infected children, who currently rely on lifelong ART. Considering the risks and expense associated with ART interruption trials, the identification of biomarkers of viral rebound will prioritize promising therapeutic intervention strategies, including anti-HIV Env protein therapeutics. However, comprehensive studies to identify those biomarkers are logistically challenging in human infants, demanding the need for relevant nonhuman primate models of HIV rebound. In this study, we developed an infant RM model of oral infection with simian-human immunodeficiency virus expressing clade C HIV Env and short-term ART followed by ATI, longitudinally characterizing the immune responses to viral infection during ART and after ATI. Additionally, we compared this infant RM model to an analogous adult RM rebound model and identified virologic and immunologic correlates of the time to viral rebound after ATI.

Analyses of human clinical HIV-1 vaccine trials and preclinical vaccine studies performed in rhesus macaque (RM) models have identified associations between non-neutralizing Fc Receptor (FcR)-dependent antibody effector functions and reduced risk of infection. Specifically, antibody-dependent phagocytosis (ADP) has emerged as a common correlate of reduced infection risk in multiple RM studies and the human HVTN505 trial. This recurrent finding suggests that antibody responses with the capability to mediate ADP are most likely a desirable component of vaccine responses aimed at protecting against HIV-1 acquisition. As use of RM models is essential for development of the next generation of candidate HIV-1 vaccines, there is a need to determine how effectively ADP activity observed in RMs translates to activity in humans. In this study we compared ADP activity of human and RM monocytes and polymorphonuclear leukocytes (PMN) to bridge this gap in knowledge. We observed considerable variability in the magnitude of monocyte and PMN ADP activity across individual humans and RM that was not dependent on FcR alleles, and only modestly impacted by cell-surface levels of FcRs. Importantly, we found that for both human and RM phagocytes, ADP activity of antibodies targeting the CD4 binding site was greatest when mediated by human IgG3, followed by RM and human IgG1. These results demonstrate that there is functional homology between antibody and FcRs from these two species for ADP. We also used novel RM IgG1 monoclonal antibodies engineered with elongated hinge regions to show that hinge elongation augments RM ADP activity. The RM IgGs with engineered hinge regions can achieve ADP activity comparable to that observed with human IgG3. These novel modified antibodies will have utility in passive immunization studies aimed at defining the role of IgG3 and ADP in protection from virus challenge or control of disease in RM models. Our results contribute to a better translation of human and macaque antibody and FcR biology, and may help to improve testing accuracy and evaluations of future active and passive prevention strategies.