Relationship of maternal cytomegalovirus-specific antibody responses and viral load to vertical transmission risk following primary maternal infection in a rhesus macaque model.

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.

FcRγ- NK Cell Induction by Specific Cytomegalovirus and Expansion by Subclinical Viral Infections in Rhesus Macaques.

"Adaptive" NK cells, characterized by FcRγ deficiency and enhanced responsiveness to Ab-bound, virus-infected cells, have been found in certain hCMV-seropositive individuals. Because humans are exposed to numerous microbes and environmental agents, specific relationships between hCMV and FcRγ-deficient NK cells (also known as g-NK cells) have been challenging to define. Here, we show that a subgroup of rhesus CMV (RhCMV)-seropositive macaques possesses FcRγ-deficient NK cells that stably persist and display a phenotype resembling human FcRγ-deficient NK cells. Moreover, these macaque NK cells resembled human FcRγ-deficient NK cells with respect to functional characteristics, including enhanced responsiveness to RhCMV-infected target in an Ab-dependent manner and hyporesponsiveness to tumor and cytokine stimulation. These cells were not detected in specific pathogen-free (SPF) macaques free of RhCMV and six other viruses; however, experimental infection of SPF animals with RhCMV strain UCD59, but not RhCMV strain 68-1 or SIV, led to induction of FcRγ-deficient NK cells. In non-SPF macaques, coinfection by RhCMV with other common viruses was associated with higher frequencies of FcRγ-deficient NK cells. These results support a causal role for specific CMV strain(s) in the induction of FcRγ-deficient NK cells and suggest that coinfection by other viruses further expands this memory-like NK cell pool.

Pathogenesis of Wild-Type-Like Rhesus Cytomegalovirus Strains following Oral Exposure of Immune-Competent Rhesus Macaques.

Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques (Macaca mulatta) is a valuable nonhuman primate model of human CMV (HCMV) persistence and pathogenesis. In vivo studies predominantly use tissue culture-adapted variants of RhCMV that contain multiple genetic mutations compared to wild-type (WT) RhCMV. In many studies, animals have been inoculated by nonnatural routes (e.g., subcutaneous, intravenous) that do not recapitulate disease progression via the normative route of mucosal exposure. Accordingly, the natural history of RhCMV would be more accurately reproduced by infecting macaques with strains of RhCMV that reflect the WT genome using natural routes of mucosal transmission. Here, we tested two WT-like RhCMV strains, UCD52 and UCD59, and demonstrated that systemic infection and frequent, high-titer viral shedding in bodily fluids occurred following oral inoculation. RhCMV disseminated to a broad range of tissues, including the central nervous system and reproductive organs. Commonly infected tissues included the thymus, spleen, lymph nodes, kidneys, bladder, and salivary glands. Histological examination revealed prominent nodular hyperplasia in spleens and variable levels of lymphoid lymphofollicular hyperplasia in lymph nodes. One of six inoculated animals had limited viral dissemination and shedding, with commensurately weak antibody responses to RhCMV antigens. These data suggest that long-term RhCMV infection parameters might be restricted by local innate factors and/or de novo host immune responses in a minority of primary infections. Together, we have established an oral RhCMV infection model that mimics natural HCMV infection. The virological and immunological parameters characterized in this study will greatly inform HCMV vaccine designs for human immunization. IMPORTANCE Human cytomegalovirus (HCMV) is globally ubiquitous with high seroprevalence rates in all communities. HCMV infections can occur vertically following mother-to-fetus transmission across the placenta and horizontally following shedding of virus in bodily fluids in HCMV-infected hosts and subsequent exposure of susceptible individuals to virus-laden fluids. Intrauterine HCMV has long been recognized as an infectious threat to fetal growth and development. Since vertical HCMV infections occur following horizontal HCMV transmission to the pregnant mother, the nonhuman primate model of HCMV pathogenesis was used to characterize the virological and immunological parameters of infection following primary mucosal exposures to rhesus cytomegalovirus.

Chest wall perforator flaps for breast reconstruction: international survey on attitudes and training needs.

BACKGROUND: Volume replacement using chest wall perforator flaps (CWPFs) is a promising technique to reduce mastectomy rates without sacrificing function or aesthetics. Owing to limited availability of the technique, only a minority of patients currently have access to CWPF procedures. METHODS: An international web-based survey was disseminated through social media, dedicated webpages, and national and international societies for breast surgery. The survey explored surgeons' attitudes towards CWPFs and their perceived training needs. RESULTS: Of 619 respondents, 88.4 per cent agreed that CWPF surgery was desirable, with one-third offering it and performing a median of 10 (i.q.r. 5-15) procedures annually. They were more likely to be senior (OR 1.35, 95 per cent c.i. 1.18 to 1.55; P < 0.001), with formal oncoplastic training (OR 4.80, 3.09 to 7.48; P < 0.001), and working in larger units (OR 1.18, 1.03 to 1.35; P = 0.018) with a free-flap (OR 1.62, 1.06 to 2.48; P = 0.025) or CWPF (OR 3.02, 1.87 to 4.89; P < 0.001) service available. In cluster and latent class analysis, none showed high cohesion with performance of CWPF surgery. CONCLUSION: There is a discrepancy between perceived importance and availability of CWPF surgery, indicating that optimal training is needed.

Horizontal transmission of endemic viruses among rhesus macaques (Macaca mulatta): Implications for human cytomegalovirus vaccine/challenge design.

INTRODUCTION: Rhesus macaques are natural hosts to multiple viruses including rhesus cytomegalovirus (RhCMV), rhesus rhadinovirus (RRV), and Simian Foamy Virus (SFV). While viral infections are ubiquitous, viral transmissions to uninfected animals are incompletely defined. Management procedures of macaque colonies include cohorts that are Specific Pathogen Free (SPF). Greater understanding of viral transmission would augment SPF protocols. Moreover, vaccine/challenge studies of human viruses would be enhanced by leveraging transmission of macaque viruses to recapitulate expected challenges of human vaccine trials. MATERIALS AND METHODS: This study characterizes viral transmissions to uninfected animals following inadvertent introduction of RhCMV/RRV/SFV-infected adults to a cohort of uninfected juveniles. Following co-housing with virus-positive adults, juveniles were serially evaluated for viral infection. RESULTS: Horizontal viral transmission was rapid and absolute, reaching 100% penetrance between 19 and 78 weeks. CONCLUSIONS: This study provides insights into viral natural histories with implications for colony management and modeling vaccine-mediated immune protection studies.

Simian homologues of human herpesviruses and implications for novel viral introduction to free-living mountain gorillas.

The endangered mountain gorilla (Gorilla beringei beringei) in Rwanda, Uganda, and the Democratic Republic of Congo is frequently in contact with humans through tourism, research activities, and illegal entry of people into protected gorilla habitat. Herpesviruses, which are ubiquitous in primates, have the potential to be shared in any setting where humans and gorillas share habitat. Based on serological findings and clinical observations of orofacial ulcerated lesions resembling herpetic lesions, an alpha-herpesvirus resembling human herpes simplex virus type 1 (HSV-1) has long been suspected to be present in human-habituated mountain gorillas in the wild. While the etiology of orofacial lesions in the wild has not been confirmed, HSV-1 has been suspected in captively-housed mountain gorillas and confirmed in a co-housed confiscated Grauer's gorilla (Gorilla beringei graueri). To better characterize herpesviruses infecting mountain gorillas and to determine the presence/absence of HSV-1 in the free-living population, we conducted a population-wide survey to test for the presence of orally shed herpesviruses. DNA was extracted from discarded chewed plants collected from 294 individuals from 26 groups, and samples were screened by polymerase chain reaction using pan-herpesvirus and HSV-1-specific assays. We found no evidence that human herpesviruses had infected free-ranging mountain gorillas. However, we found gorilla-specific homologs to human herpesviruses, including cytomegaloviruses (GbbCMV-1 and 2), a lymphocryptovirus (GbbLCV-1), and a new rhadinovirus (GbbRHV-1) with similar characteristics (i.e., timing of primary infection, shedding in multiple age groups, and potential modes of transmission) to their human counterparts, human cytomegalovirus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, respectively.

Horizontal Transmission of Cytomegalovirus in a Rhesus Model Despite High-Level, Vaccine-Elicited Neutralizing Antibody and T-Cell Responses.

The development of a vaccine to prevent congenital human cytomegalovirus (HCMV) disease is a public health priority. We tested rhesus CMV (RhCMV) prototypes of HCMV vaccine candidates in a seronegative macaque oral challenge model. Immunogens included a recombinant pentameric complex (PC; gH/gL/pUL128/pUL130/pUL131A), a postfusion gB ectodomain, and a DNA plasmid that encodes pp65-2. Immunization with QS21-adjuvanted PC alone or with the other immunogens elicited neutralizing titers comparable to those elicited by RhCMV infection. Similarly, immunization with all 3 immunogens elicited pp65-specific cytotoxic T-cell responses comparable to those elicited by RhCMV infection. RhCMV readily infected immunized animals and was detected in saliva, blood, and urine after challenge in quantities similar to those in placebo-immunized animals. If HCMV evades vaccine-elicited immunity in humans as RhCMV evaded immunity in macaques, a HCMV vaccine must elicit immunity superior to, or different from, that elicited by the prototype RhCMV vaccine to block horizontal transmission.

Intrahost cytomegalovirus population genetics following antibody pretreatment in a monkey model of congenital transmission.

Human cytomegalovirus (HCMV) infection is the leading non-genetic cause of congenital birth defects worldwide. While several studies have addressed the genetic composition of viral populations in newborns diagnosed with HCMV, little is known regarding mother-to-child viral transmission dynamics and how therapeutic interventions may impact within-host viral populations. Here, we investigate how preexisting CMV-specific antibodies shape the maternal viral population and intrauterine virus transmission. Specifically, we characterize the genetic composition of CMV populations in a monkey model of congenital CMV infection to examine the effects of passively-infused hyperimmune globulin (HIG) on viral population genetics in both maternal and fetal compartments. In this study, 11 seronegative, pregnant monkeys were challenged with rhesus CMV (RhCMV), including a group pretreated with a standard potency HIG preparation (n = 3), a group pretreated with a high-neutralizing potency HIG preparation (n = 3), and an untreated control group (n = 5). Targeted amplicon deep sequencing of RhCMV glycoprotein B and L genes revealed that one of the three strains present in the viral inoculum (UCD52) dominated maternal and fetal viral populations. We identified minor haplotypes of this strain and characterized their dynamics. Many of the identified haplotypes were consistently detected at multiple timepoints within sampled maternal tissues, as well as across tissue compartments, indicating haplotype persistence over time and transmission between maternal compartments. However, haplotype numbers and diversity levels were not appreciably different between control, standard-potency, and high-potency pretreatment groups. We found that while the presence of maternal antibodies reduced viral load and congenital infection, it had no apparent impact on intrahost viral genetic diversity at the investigated loci. Interestingly, some minor haplotypes present in fetal and maternal-fetal interface tissues were also identified as minor haplotypes in corresponding maternal tissues, providing evidence for a loose RhCMV mother-to-fetus transmission bottleneck even in the presence of preexisting antibodies.

In vitro and in vivo characterization of a recombinant rhesus cytomegalovirus containing a complete genome.

Cytomegaloviruses (CMVs) are highly adapted to their host species resulting in strict species specificity. Hence, in vivo examination of all aspects of CMV biology employs animal models using host-specific CMVs. Infection of rhesus macaques (RM) with rhesus CMV (RhCMV) has been established as a representative model for infection of humans with HCMV due to the close evolutionary relationships of both host and virus. However, the only available RhCMV clone that permits genetic modifications is based on the 68-1 strain which has been passaged in fibroblasts for decades resulting in multiple genomic changes due to tissue culture adaptations. As a result, 68-1 displays reduced viremia in RhCMV-naïve animals and limited shedding compared to non-clonal, low passage isolates. To overcome this limitation, we used sequence information from primary RhCMV isolates to construct a full-length (FL) RhCMV by repairing all mutations affecting open reading frames (ORFs) in the 68-1 bacterial artificial chromosome (BAC). Inoculation of adult, immunocompetent, RhCMV-naïve RM with the reconstituted virus resulted in significant viremia in the blood similar to primary isolates of RhCMV and furthermore led to high viral genome copy numbers in many tissues at day 14 post infection. In contrast, viral dissemination was greatly reduced upon deletion of genes also lacking in 68-1. Transcriptome analysis of infected tissues further revealed that chemokine-like genes deleted in 68-1 are among the most highly expressed viral transcripts both in vitro and in vivo consistent with an important immunomodulatory function of the respective proteins. We conclude that FL-RhCMV displays in vitro and in vivo characteristics of a wildtype virus while being amenable to genetic modifications through BAC recombineering techniques.

Bridging the gap: Using reservoir ecology and human serosurveys to estimate Lassa virus spillover in West Africa.

Forecasting the risk of pathogen spillover from reservoir populations of wild or domestic animals is essential for the effective deployment of interventions such as wildlife vaccination or culling. Due to the sporadic nature of spillover events and limited availability of data, developing and validating robust, spatially explicit, predictions is challenging. Recent efforts have begun to make progress in this direction by capitalizing on machine learning methodologies. An important weakness of existing approaches, however, is that they generally rely on combining human and reservoir infection data during the training process and thus conflate risk attributable to the prevalence of the pathogen in the reservoir population with the risk attributed to the realized rate of spillover into the human population. Because effective planning of interventions requires that these components of risk be disentangled, we developed a multi-layer machine learning framework that separates these processes. Our approach begins by training models to predict the geographic range of the primary reservoir and the subset of this range in which the pathogen occurs. The spillover risk predicted by the product of these reservoir specific models is then fit to data on realized patterns of historical spillover into the human population. The result is a geographically specific spillover risk forecast that can be easily decomposed and used to guide effective intervention. Applying our method to Lassa virus, a zoonotic pathogen that regularly spills over into the human population across West Africa, results in a model that explains a modest but statistically significant portion of geographic variation in historical patterns of spillover. When combined with a mechanistic mathematical model of infection dynamics, our spillover risk model predicts that 897,700 humans are infected by Lassa virus each year across West Africa, with Nigeria accounting for more than half of these human infections.

Neutralization of rhesus cytomegalovirus IL-10 reduces horizontal transmission and alters long-term immunity.

Human cytomegalovirus (HCMV) causes severe disease in infants and immunocompromised people. There is no approved HCMV vaccine, and vaccine development strategies are complicated by evidence of both persistent infection and reinfection of people with prior immunity. The greatest emphasis has been placed on reducing transmission to seronegative pregnant women to prevent vertical transmission and its potentially severe sequelae. Increasing evidence suggests that the earliest host-HCMV interactions establish conditions for viral persistence, including evasion of host immune responses to the virus. Using a nonhuman primate model of HCMV infection, we show that rhesus macaques immunized against viral interleukin-10 (IL-10) manifest delayed rhesus cytomegalovirus (RhCMV) acquisition and altered immune responses to the infection when it does occur. Among animals with the greatest antiviral IL-10-neutralizing activity, the timing of RhCMV seroconversion was delayed by an average of 12 weeks. After acquisition, such animals displayed an antibody response to the new infection, which peaked as expected after 2 weeks but then declined rapidly. In contrast, surprisingly, vaccination with glycoprotein B (gB) protein had no discernible impact on these outcomes. Our results demonstrate that viral IL-10 is a key regulator of successful host immune responses to RhCMV. Viral IL-10 is, therefore, an important target for vaccine strategies against cytomegalovirus (CMV). Furthermore, given the immunoregulatory function of viral IL-10, targeting this protein may prove synergistic with other vaccine therapies and targets. Our study also provides additional evidence that the earliest host-CMV interactions can have a significant impact on the nature of persistent infection.

Real-time ex vivo perfusion of human lymph nodes invaded by cancer (REPLICANT): a feasibility study.

Understanding how breast cancer (BC) grows in axillary lymph nodes (ALNs), and refining how therapies might halt that process, is clinically important. However, modelling the complex ALN microenvironment is difficult, and no human models exist at present. We harvested ALNs from ten BC patients, and perfused them at 37 °C ex vivo for up to 24 h. Controlled autologous testing showed that ALNs remain viable after 24 h of ex vivo perfusion: haematoxylin and eosin-stained histological appearance and proliferation (by Ki67 immunohistochemistry) did not change significantly over time for any perfused ALN compared with a control from time-point zero. Furthermore, targeted gene expression analysis (NanoString PanCancer IO360 panel) showed that only 21/750 genes were differentially expressed between control and perfused ALNs (|log2 FC| > 1 and q < 0.1): none were involved in apoptosis and metabolism, but rather all 21 genes were involved in immune function and angiogenesis. During perfusion, tissue acid-base balance remained stable. Interestingly, the flow rate increased (p < 0.001) in cancer-replaced (i.e. metastasis occupied more than 90% of the surface area on multiple levels) compared to cancer-free nodes (i.e. nodes with no metastasis on multiple sections). CXCL11 transcripts were significantly more abundant in cancer-replaced nodes, while CXCL12 transcripts were significantly more abundant in cancer-free nodes. These cytokines were also detected in the circulating perfusate. Monoclonal antibodies (nivolumab and trastuzumab) were administered into a further three ALNs to confirm perfusion efficacy. These drugs saturated the nodes; nivolumab even induced cancer cell death. Normothermic ALN perfusion is not only feasible but sustains the tumour microenvironment ex vivo for scientific investigation. This model could facilitate the identification of actionable immuno-oncology targets. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Subclinical Cytomegalovirus Infection Is Associated with Altered Host Immunity, Gut Microbiota, and Vaccine Responses.

Subclinical viral infections (SVI), including cytomegalovirus (CMV), are highly prevalent in humans, resulting in lifelong persistence. However, the impact of SVI on the interplay between the host immunity and gut microbiota in the context of environmental exposures is not well defined. We utilized the preclinical nonhuman primate (NHP) model consisting of SVI-free (specific-pathogen-free [SPF]) rhesus macaques and compared them to the animals with SVI (non-SPF) acquired through natural exposure and investigated the impact of SVI on immune cell distribution and function, as well as on gut microbiota. These changes were examined in animals housed in the outdoor environment compared to the controlled indoor environment. We report that SVI are associated with altered immune cell subsets and gut microbiota composition in animals housed in the outdoor environment. Non-SPF animals harbored a higher proportion of potential butyrate-producing Firmicutes and higher numbers of lymphocytes, effector T cells, and cytokine-producing T cells. Surprisingly, these differences diminished following their transfer to the controlled indoor environment, suggesting that non-SPFs had increased responsiveness to environmental exposures. An experimental infection of indoor SPF animals with CMV resulted in an increased abundance of butyrate-producing bacteria, validating that CMV enhanced colonization of butyrate-producing commensals. Finally, non-SPF animals displayed lower antibody responses to influenza vaccination compared to SPF animals. Our data show that subclinical CMV infection heightens host immunity and gut microbiota changes in response to environmental exposures. This may contribute to the heterogeneity in host immune response to vaccines and environmental stimuli at the population level.IMPORTANCE Humans harbor several latent viruses that modulate host immunity and commensal microbiota, thus introducing heterogeneity in their responses to pathogens, vaccines, and environmental exposures. Most of our understanding of the effect of CMV on the immune system is based on studies of children acquiring CMV or of immunocompromised humans with acute or reactivated CMV infection or in ageing individuals. The experimental mouse models are genetically inbred and are completely adapted to the indoor laboratory environment. In contrast, nonhuman primates are genetically outbred and are raised in the outdoor environment. Our study is the first to report the impact of long-term subclinical CMV infection on host immunity and gut microbiota, which is evident only in the outdoor environment but not in the indoor environment. The significance of this study is in highlighting the impact of SVI on enhancing host immune susceptibility to environmental exposures and immune heterogeneity.

Suspected Exposure to Filoviruses Among People Contacting Wildlife in Southwestern Uganda.

Background: Human and filovirus host interactions remain poorly understood in areas where Ebola hemorrhagic fever outbreaks are likely to occur. In the Bwindi region of Uganda, a hot spot of mammalian biodiversity in Africa, human livelihoods are intimately connected with wildlife, creating potential for exposure to filoviruses. Methods: We tested samples from 331 febrile patients presenting to healthcare facilities near Bwindi Impenetrable Forest, Uganda, by polymerase chain reaction (PCR) analysis and Western blot, using recombinant glycoprotein antigens for Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus. Behavioral data on contact with wildlife were collected to examine risk factors for filovirus seropositivity. Results: All patients were negative for active filovirus infection, by PCR analysis. However, patients were seroreactive to SUDV (4.7%), EBOV (5.3%), and BDBV (8.9%), indicating previous exposure. Touching duikers was the most significant risk factor associated with EBOV seropositivity, while hunting primates and touching and/or eating cane rats were significant risk factors for SUDV seropositivity. Conclusions: People in southwestern Uganda have suspected previous exposure to filoviruses, particularly those with a history of wildlife contact. Circulation of filoviruses in wild animals and subsequent spillover into humans could be more common than previously reported.

Validation of the Vectra XT three-dimensional imaging system for measuring breast volume and symmetry following oncological reconstruction.

PURPOSE: Three-dimensional surface imaging (3D-SI) of the breasts enables the measurement of breast volume and shape symmetry. If these measurements were sufficiently accurate and repeatable, they could be used in planning oncological breast surgery and as an objective measure of aesthetic outcome. The aim of this study was to validate the measurements of breast volume and symmetry provided by the Vectra XT imaging system. METHODS: To validate measurements, breast phantom models of true volume between 100 and 1000 cm3 were constructed and varying amounts removed to mimic breast tissue 'resections'. The volumes of the phantoms were measured using 3D-SI by two observers and compared to a gold standard. For intra-observer repeatability and inter-observer reproducibility in vivo, 16 patients who had undergone oncological breast surgery had breast volume and symmetry measured three times by two observers. RESULTS: A mean relative difference of 2.17 and 2.28% for observer 1 and 2 respectively was seen in the phantom measurements compared to the gold standard (n = 45, Bland Altman agreement). Intra-observer variation over ten repeated measurements demonstrated mean coefficients of variation (CV) of 0.58 and 0.49%, respectively. The inter-observer variation demonstrated a mean relative difference of 0.11% between the two observers. In patients, intra-observer variation over three repeated volume measurements for each observer was 3.9 and 3.8% (mean CV); the mean relative difference between observers was 5.78%. For three repeated shape symmetry measurements using RMS projection difference between the two breasts, the intra-observer variations were 8 and 14% (mean CV), the mean relative difference between observers was 0.43 mm for average symmetry values that ranged from about 3.5 to 15.5 mm. CONCLUSION: This first validation of breast volume and shape symmetry measurements using the Vectra XT 3D-SI system suggests that these measurements have the potential to assist in pre-operative planning and also as a measure of aesthetic outcome.

Identification of a Continuous Neutralizing Epitope within UL128 of Human Cytomegalovirus.

As human cytomegalovirus (HCMV) is the most common infectious cause of fetal anomalies during pregnancy, development of a vaccine that prevents HCMV infection is considered a global health priority. Although HCMV immune correlates of protection are only poorly defined, neutralizing antibodies (NAb) targeting the envelope pentamer complex (PC) composed of the subunits gH, gL, UL128, UL130, and UL131A are thought to contribute to the prevention of HCMV infection. Here, we describe a continuous target sequence within UL128 that is recognized by a previously isolated potent PC-specific NAb termed 13B5. By using peptide-based scanning procedures, we identified a 13-amino-acid-long target sequence at the UL128 C terminus that binds the 13B5 antibody with an affinity similar to that of the purified PC. In addition, the 13B5 binding site is universally conserved in HCMV, contains a previously described UL128/gL interaction site, and interferes with the 13B5 neutralizing function, indicating that the 13B5 epitope sequence is located within the PC at a site of critical importance for HCMV neutralization. Vaccination of mice with peptides containing the 13B5 target sequence resulted in the robust stimulation of binding antibodies and, in a subset of immunized animals, in the induction of detectable NAb, supporting that the identified 13B5 target sequence constitutes a PC-specific neutralizing epitope. These findings provide evidence for the discovery of a continuous neutralizing epitope within the UL128 subunit of the PC that could be an important target of humoral immune responses that are involved in protection against congenital HCMV infection.IMPORTANCE Neutralizing antibodies (NAb) targeting the human cytomegalovirus (HCMV) envelope pentamer complex (PC) are thought to be important for preventing HCMV transmission from the mother to the fetus, thereby mitigating severe developmental disabilities in newborns. However, the epitope sequences within the PC that are recognized by these potentially protective antibody responses are only poorly defined. Here, we provide evidence for the existence of a highly conserved, continuous, PC-specific epitope sequence that appears to be located within the PC at a subunit interaction site of critical importance for HCMV neutralization. These discoveries provide insights into a continuous PC-specific neutralizing epitope, which could be an important target for a vaccine formulation to interfere with congenital HCMV infection.

Maternal CD4+ T cells protect against severe congenital cytomegalovirus disease in a novel nonhuman primate model of placental cytomegalovirus transmission.

Elucidation of maternal immune correlates of protection against congenital cytomegalovirus (CMV) is necessary to inform future vaccine design. Here, we present a novel rhesus macaque model of placental rhesus CMV (rhCMV) transmission and use it to dissect determinants of protection against congenital transmission following primary maternal rhCMV infection. In this model, asymptomatic intrauterine infection was observed following i.v. rhCMV inoculation during the early second trimester in two of three rhCMV-seronegative pregnant females. In contrast, fetal loss or infant CMV-associated sequelae occurred in four rhCMV-seronegative pregnant macaques that were CD4(+) T-cell depleted at the time of inoculation. Animals that received the CD4(+) T-cell-depleting antibody also exhibited higher plasma and amniotic fluid viral loads, dampened virus-specific CD8(+) T-cell responses, and delayed production of autologous neutralizing antibodies compared with immunocompetent monkeys. Thus, maternal CD4(+) T-cell immunity during primary rhCMV infection is important for controlling maternal viremia and inducing protective immune responses that prevent severe CMV-associated fetal disease.

The potential role of three-dimensional surface imaging as a tool to evaluate aesthetic outcome after Breast Conserving Therapy (BCT).

PURPOSE: To establish whether objective measurements of symmetry of volume and shape using three-dimensional surface imaging (3D-SI) can be used as surrogate markers of aesthetic outcome in patients who have undergone breast conserving therapy (BCT). METHODS: Women who had undergone unilateral BCT in the preceding 1-6 years were invited to participate. Participants completed a satisfaction questionnaire (BREAST-Q) and underwent 3D-SI. Volume and surface symmetry were measured on the images. Assessment of aesthetic outcome was undertaken by a panel of clinicians. The Kruskal-Wallis test was used to assess the relationship between volume and shape symmetry measurements with the panel score. Spearman's rho correlations were used to assess the relationship between the measurements and patient satisfaction. RESULTS: 200 women participated. Median volume symmetry was 87% (IQR 78-93) and shape symmetry was 5.9 mm (IQR 4.2-8.0). The participants were grouped according to panel assessment of aesthetic outcome (poor, fair, good, excellent) and the median volume and shape symmetry was calculated for each group. Volume symmetry significantly differed between the groups. Post hoc pairwise comparisons demonstrated that these differences existed between panel scores of fair versus good and good versus excellent. Median shape symmetry also differed according to patient panel groups with four significant pairwise comparisons between poor versus good, poor versus excellent, fair versus good and fair versus excellent. There was a significant but weak correlation of both volume symmetry and surface asymmetry with BREAST-Q scores (correlation coefficients 0.187 and -0.229, respectively). CONCLUSION: Breast volume and shape symmetry are both associated with panel assessment scores and patient satisfaction. The objective volume and shape symmetry measures were strongly associated with panel assessment scores, such that a 3D-SI tool could replace panel assessment as a faster and more objective method of evaluating aesthetic outcomes.

Exploitation of Interleukin-10 (IL-10) Signaling Pathways: Alternate Roles of Viral and Cellular IL-10 in Rhesus Cytomegalovirus Infection.

There is accumulating evidence that the viral interleukin-10 (vIL-10) ortholog of both human and rhesus cytomegalovirus (HCMV and RhCMV, respectively) suppresses the functionality of cell types that are critical to contain virus dissemination and help shape long-term immunity during the earliest virus-host interactions. In particular, exposure of macrophages, peripheral blood mononuclear cells, monocyte-derived dendritic cells, and plasmacytoid dendritic cells to vIL-10 suppresses multiple effector functions including, notably, those that link innate and adaptive immune responses. Further, vaccination of RhCMV-uninfected rhesus macaques with nonfunctional forms of RhCMV vIL-10 greatly restricted parameters of RhCMV infection following RhCMV challenge of the vaccinees. Vaccinees exhibited significantly reduced shedding of RhCMV in saliva and urine following RhCMV challenge compared to shedding in unvaccinated controls. Based on the evidence that vIL-10 is critical during acute infection, the role of vIL-10 during persistent infection was analyzed in rhesus macaques infected long term with RhCMV to determine whether postinfection vaccination against vIL-10 could change the virus-host balance. RhCMV-seropositive macaques, which shed RhCMV in saliva, were vaccinated with nonfunctional RhCMV vIL-10, and shedding levels of RhCMV in saliva were evaluated. Following robust increases in vIL-10-binding and vIL-10-neutralizing antibodies, shedding levels of RhCMV modestly declined, consistent with the interpretation that vIL-10 may play a functional role during persistent infection. However, a more significant association was observed between the levels of cellular IL-10 secreted in peripheral blood mononuclear cells exposed to RhCMV antigens and shedding of RhCMV in saliva. This result implies that RhCMV persistence is associated with the induction of cellular IL-10 receptor-mediated signaling pathways. IMPORTANCE: Human health is adversely impacted by viruses that establish lifelong infections that are often accompanied with increased morbidity and mortality (e.g., infections with HIV, hepatitis C virus, or human cytomegalovirus). A longstanding but unfulfilled goal has been to develop postinfection vaccine strategies that could "reboot" the immune system of an infected individual in ways that would enable the infected host to develop immune responses that clear reservoirs of persistent virus infection, effectively curing the host of infection. This concept was evaluated in rhesus macaques infected long term with rhesus cytomegalovirus by repeatedly immunizing infected animals with nonfunctional versions of the rhesus cytomegalovirus-encoded viral interleukin-10 immune-modulating protein. Following vaccine-mediated boosting of antibody titers to viral interleukin-10, there was modest evidence for increased immunological control of the virus following vaccination. More significantly, data were also obtained that indicated that rhesus cytomegalovirus is able to persist due to upregulation of the cellular interleukin-10 signaling pathway.

The susceptibility of primary cultured rhesus macaque kidney epithelial cells to rhesus cytomegalovirus strains.

Kidney epithelial cells are common targets for human and rhesus cytomegalovirus (HCMV and RhCMV) in vivo, and represent an important reservoir for long-term CMV shedding in urine. To better understand the role of kidney epithelial cells in primate CMV natural history, primary cultures of rhesus macaque kidney epithelial cells (MKE) were established and tested for infectivity by five RhCMV strains, including two wild-type strains (UCD52 and UCD59) and three strains containing different coding contents in UL/b'. The latter strains included 180.92 [containing an intact RhUL128-RhUL130-R hUL131 (RhUL128L) locus but deleted for the UL/b' RhUL148-rh167-loci], 68-1 (RhUL128L-defective and fibroblast-tropic) and BRh68-1.2 (the RhUL128L-repaired version of 68-1). As demonstrated by RhCMV cytopathic effect, plaque formation, growth kinetics and early virus entry, we showed that MKE were differentially susceptible to RhCMV infection, related to UL/b' coding contents of the different strains. UCD52 and UCD59 replicated vigorously in MKE, 68-1 replicated poorly, and 180.92 grew with intermediate kinetics. Reconstitution of RhUL128L in 68-1 (BRh68-1.2) restored its replication efficiency in MKE as compared to UCD52 and UCD59, consistent with the essential role of UL128L for HCMV epithelial tropism. Further analysis revealed that the UL/b' UL148-rh167-loci deletion in 180.92 impaired RhUL132 (rh160) expression. Given that 180.92 retains an intact RhUL128L, but genetically or functionally lacks genes from RhUL132 (rh160) to rh167 in UL/b', its attenuated infection efficiency indicated that, along with RhUL128L, an additional protein(s) encoded within the UL/b' RhUL132 (rh160)-rh167 region (potentially, RhUL132 and/or RhUL148) is indispensable for efficient replication in MKE.