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We examined the long-term causal effects of an evidence-based parenting program delivered in infancy on children's emotion regulation and resting-state functional connectivity (rs-fc) during middle childhood. Families were referred to the study by Child Protective Services (CPS) as part of a diversion from a foster care program. A low-risk group of families was also recruited. CPS-involved families were randomly assigned to receive the target (Attachment and Biobehavioral Catch-up, ABC) or a control intervention (Developmental Education for Families, DEF) before infants turned 2. Both interventions were home-based, manualized, and 10-sessions long. During middle childhood, children underwent a 6-min resting-state functional MRI scan. Amygdala seed-based rs-fc analysis was completed with intervention group as the group-level predictor of interest. Fifty-seven children ( N ABC = 21; N DEF = 17; N COMP = 19; M age = 10.02 years, range = 8.08-12.14) were scanned successfully. The DEF group evidenced negative left amygdala↔OFC connectivity, whereas connectivity was near zero in the ABC and comparison groups (ABCvsDEF: Cohen's d = 1.17). ABC may enhance high-risk children's regulatory neurobiology outcomes ∼8 years after the intervention was completed.

Objective: Early adverse parenting predicts various negative outcomes, including psychopathology and altered development. Animal work suggests that adverse parenting might change amygdala-prefrontal cortex (PFC) circuitry, but work in humans remains correlational. The present study leveraged data from a randomized controlled trial examining the efficacy of an early parenting intervention targeting parental nurturance and sensitivity (Attachment and Biobehavioral Catch-up [ABC]) to test whether early parenting quality causally affects amygdala-PFC connectivity later in life., Method: Participants (N = 60, mean age = 10.0 years) included 41 high-risk children whose parents were referred by Child Protective Services and randomly assigned to receive either ABC (n = 21) or a control intervention (n = 20) during the children's infancy and a comparison sample of low-risk children (n = 19). Amygdala-PFC connectivity was assessed via functional magnetic resonance imaging while children viewed fearful and neutral faces., Results: Across facial expressions, ABC produced different changes than the control intervention in amygdala-PFC connectivity in response to faces. The ABC group also exhibited greater responses than the control intervention group to faces in areas classically associated with emotion regulation, including the orbitofrontal cortex and right insula. Mediation analysis suggested that the effect of ABC on PFC activation was mediated by the intervention's effect on amygdala-PFC connectivity., Conclusion: Results provide preliminary causal evidence for the effect of early parenting intervention on amygdala-PFC connectivity and on PFC responses to face viewing. Findings also highlight amygdala-PFC connectivity as a potential mediator of the effects of early parenting intervention on children's emotion regulation development., Clinical Trial Registration Information: Intervening Early With Neglected Children; https://clinicaltrials.gov/; NCT02093052., Diversity & Inclusion Statement: We worked to ensure sex and gender balance in the recruitment of human participants. We worked to ensure race, ethnic, and/or other types of diversity in the recruitment of human participants. We worked to ensure that the study questionnaires were prepared in an inclusive way. One or more of the authors of this paper self-identifies as a member of one or more historically underrepresented racial and/or ethnic groups in science. One or more of the authors of this paper self-identifies as a member of one or more historically underrepresented sexual and/or gender groups in science. One or more of the authors of this paper received support from a program designed to increase minority representation in science. While citing references scientifically relevant for this work, we also actively worked to promote sex and gender balance in our reference list., (Copyright © 2023 American Academy of Child and Adolescent Psychiatry. All rights reserved.)

Positive associations have been found between cortical thickness and measures of parasympathetic cardiac control (e.g., respiratory sinus arrhythmia, RSA) in adults, which may indicate mechanistic integration between neural and physiological indicators of stress regulation. However, it is unknown when in development this brain-body association arises and whether the direction of association and neuroanatomical localization vary across development. To investigate this, we collected structural magnetic resonance imaging and resting-state respiratory sinus arrhythmia data from children in middle childhood (N = 62, M age  = 10.09, range: 8.28-12.14 years). Whole-brain and exploratory ROI analyses revealed positive associations between RSA and cortical thickness in four frontal and parietal clusters in the left hemisphere and one cluster in the right. Exploratory ROI analyses revealed a similar positive association between cortical thickness and RSA, with two regions surviving multiple comparison correction, including the inferior frontal orbital gyrus and the Sylvian fissure. Prior work has identified these cortical areas as part of the central autonomic network that supports integrative regulation of stress response (e.g., autonomic, endocrine, and behavioral) and emotional expression. Our results suggest that the association between cortical thickness and resting RSA is present in middle childhood and is similar to the associations seen during adulthood. Future studies should investigate associations between RSA and cortical thickness among young children and adolescents., (© 2023 Society for Psychophysiological Research.)

Herpes simplex virus 2 (HSV-2) causes most sexually transmitted genital ulcerative disease. No effective prophylactic vaccine is currently available. Replication-defective (ICP8-) HSV stimulates immune responses in animals without producing progeny virus, making it potentially useful as a safe form of a live vaccine against HSV. We previously demonstrated that mice generate a stronger response to ICP8- virus encoding B7-2 costimulation molecules than to the parental replication-defective virus. We have also demonstrated enhanced immunogenicity of an ICP8-, virion host shutoff (vhs)- virus which can no longer destabilize viral and host mRNAs. Here, we constructed a triple mutant, ICP8-vhs-B7-2+ strain, and compared it to both double mutant viruses. Immunization of mice with a single dose of ICP8-B7-2+ or ICP8-vhs-B7-2+ virus decreased challenge virus replication in the vaginal mucosa, genital disease, and mortality more effectively than immunization with the ICP8-vhs- virus. Immunization with ICP8-B7-2+ or ICP8-vhs-B7-2+ virus also effectively suppressed subsequent HSV-2 infection of the nervous system compared to immunization with the ICP8-vhs- virus. ICP8-B7-2+ and ICP8-vhs-B7-2+ strains induced more IFN gamma-producing CD8 T cells and memory CD8 T cells than did ICP8-vhs- virus, potentially explaining the enhanced protective effects. Thus, B7 costimulation molecules expressed from a replication-defective vaccine can enhance vaccine efficacy, even in an immunocompetent host.

A variety of childhood experiences can lead to anxious/depressed (A/D) symptoms. The aim of the present study was to explore the brain morphological (cortical thickness and surface area) correlates of A/D symptoms and the extent to which these phenotypes vary depending on the quality of the parenting context in which children develop. Structural magnetic resonance imaging (MRI) scans were acquired on 45 children with Child Protective Services (CPS) involvement due to risk of not receiving adequate care (high-risk group) and 25 children without CPS involvement (low-risk group) (range age = 8.08-12.14; M age = 10.05) to assess cortical thickness (CT) and cortical surface area (SA). A/D symptoms were measured using the Child Behavioral Checklist. The association between A/D symptoms and CT, but not SA, differed by risk status such that high-risk children showed decreasing CT as A/D scores increased, whereas low-risk children showed increasing CT as A/D scores increased. This interaction was specific to CT in prefrontal, frontal, temporal, and parietal cortical regions. The groups had marginally different A/D scores, in the direction of higher risk being associated with lower A/D scores. Results suggest that CT correlates of A/D symptoms are differentially shaped by the quality of early caregiving experiences and should be distinguished between high- and low-risk children.

Fetal, infant, and toddler neuroimaging is commonly thought of as a development of modern times (last two decades). Yet, this field mobilized shortly after the discovery and implementation of MRI technology. Here, we provide a review of the parallel advancements in the fields of fetal, infant, and toddler neuroimaging, noting the shifts from clinical to research use, and the ongoing challenges in this fast-growing field. We chronicle the pioneering science of fetal, infant, and toddler neuroimaging, highlighting the early studies that set the stage for modern advances in imaging during this developmental period, and the large-scale multi-site efforts which ultimately led to the explosion of interest in the field today. Lastly, we consider the growing pains of the community and the need for an academic society that bridges expertise in developmental neuroscience, clinical science, as well as computational and biomedical engineering, to ensure special consideration of the vulnerable mother-offspring dyad (especially during pregnancy), data quality, and image processing tools that are created, rather than adapted, for the young brain., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Art exposure can influence children's emotional growth, but little is known about tools that aid emotional development in art museums. We implemented attentional and social manipulations to test whether (1) modifications to unscripted instructions and (2) caregiver prompts shape children's attentional focus towards either the emotional or elemental content (e.g., colour and medium) of paintings. These manipulations occurred within an on-going art museum education programme. Afterwards, children's (N = 60; ages 3-13 years) attentional focus towards emotions or elements was assessed by asking them to select words that best described the art. Children focused on emotion more, but the instructional manipulation successfully influenced word choices towards the targeted focus. Caregiver prompts also influenced focus towards the elements and away from emotions. These findings highlight that children's attention to art's emotional content can be altered by social context, which here was demonstrated within a museum programme., (© 2021 The British Psychological Society.)

Given that human infants are almost fully reliant on caregivers for survival, the presence of parents who provide sensitive, responsive care support infants and young children in developing the foundation for optimal biological functioning. Conversely, when parents are unavailable or insensitive, there are consequences for infants' and children's attachment and neurobiological development. In this paper, we describe effects of inadequate parenting on children's neurobiological and behavioral development, with a focus on developing capacities for executive functioning, emotion regulation, and other important cognitive-affective processes. Most prior research has examined correlational associations among these constructs. Given that interventions tested through randomized clinical trials allow for causal inferences, we review longitudinal intervention effects on children's biobehavioral and cognitive-affective outcomes. In particular, we provide an overview of the Bucharest Early Intervention Project, a study in which children were randomized to continue in orphanage care (typically the most extreme condition of privation) or were placed into the homes of trained, supported foster parents. We also discuss findings regarding Attachment and Biobehavioral Catch-up, an intervention enhancing sensitivity among high-risk parents. We conclude by suggesting future directions for research in this area., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The field of adult neuroimaging relies on well-established principles in research design, imaging sequences, processing pipelines, as well as safety and data collection protocols. The field of infant magnetic resonance imaging, by comparison, is a young field with tremendous scientific potential but continuously evolving standards. The present article aims to initiate a constructive dialog between researchers who grapple with the challenges and inherent limitations of a nascent field and reviewers who evaluate their work. We address 20 questions that researchers commonly receive from research ethics boards, grant, and manuscript reviewers related to infant neuroimaging data collection, safety protocols, study planning, imaging sequences, decisions related to software and hardware, and data processing and sharing, while acknowledging both the accomplishments of the field and areas of much needed future advancements. This article reflects the cumulative knowledge of experts in the FIT'NG community and can act as a resource for both researchers and reviewers alike seeking a deeper understanding of the standards and tradeoffs involved in infant neuroimaging., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Although antiretroviral therapy (ART) successfully suppresses HIV-1 replication, ART-treated individuals must maintain therapy to avoid rebound from an integrated viral reservoir. Strategies to limit or clear this reservoir are urgently needed. Individuals infected for longer periods prior to ART appear to harbor more genetically diverse virus, but the roles of duration of infection and viral diversity in the humoral immune response remain to be studied. We aim to clarify a role, if any, for autologous and heterologous antibodies in multi-pronged approaches to clearing infection. To that end, we have characterized the breadths and potencies of antibody responses in individuals with varying durations of infection and HIV-1 envelope ( env) gene diversity as well as the sensitivity of their inducible virus reservoir to broadly neutralizing antibodies (bNAbs). Plasma was collected from 8 well-characterized HIV-1 + males on ART with varied durations of active infection. HIV envs from reservoir-derived outgrowth viruses were amplified and single genome sequenced in order to measure genetic diversity in each participant. IgG from plasma was analyzed for binding titers against gp41 and gp120 proteins, and for neutralizing titers against a global HIV-1 reference panel as well as autologous outgrowth viruses. The sensitivity to bNAbs of these same autologous viruses was measured. Overall, we observed that greater env diversity was associated with higher neutralizing titers against the global panel and also increased resistance to certain bNAbs. Despite the presence of robust anti-HIV-1 antibody titers, we did not observe potent neutralization against autologous viruses. In fact, 3 of 8 participants harbored viruses that were completely resistant to the highest tested concentration of autologous IgG. That this lack of neutralization was observed regardless of ART duration or viral diversity suggests that the inducible reservoir harbors 'escaped' viruses (that co-evolved with autologous antibody responses), rather than proviruses archived from earlier in infection. Finally, we observed that viruses resistant to autologous neutralization remained sensitive to bNAbs, especially CD4bs and MPER bNAbs. Overall, our data suggest that the inducible reservoir is relatively resistant to autologous antibodies and that individuals with limited virus variation in the env gene, such as those who start ART early in infection, are more likely to be sensitive to bNAb treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wilson, Shakhtour, Ward, Ren, Recarey, Stevenson, Korom, Kovacs, Benko, Jones and Lynch.)

Children at risk for neglect or abuse are vulnerable to delays in inhibitory control development. Prior findings suggest that early parenting interventions that target parental sensitivity and responsiveness during infancy can improve executive function outcomes of high-risk children during preschool years; however, little is known about how persistent these gains are through middle childhood. Participants included 76 CPS-involved children who were randomly assigned to either the ABC intervention (N = 32) or the Developmental Education for Families (DEF) control intervention (N = 44), and 53 low-risk children. Children completed the Stop Signal Reaction Time (SSRT) paradigm at ages 8 and 10. Intervention group predicted performance on the SSRT at age 8 such that children who received the ABC intervention and children in the low-risk group performed significantly better than children who received the DEF intervention (ABC vs. DEF: Cohen's d = 0.92; low-risk group vs. DEF: d = 0.56). The performances of the ABC and the low-risk groups were not statistically different. There were no significant group differences in SSRT performance at age 10. These findings demonstrate that the ABC intervention has long-term beneficial effects on inhibitory control development in children with a history of early caregiving adversity. A video abstract of this article can be viewed at https://youtu.be/P9oLyfo7pYA., (© 2020 John Wiley & Sons Ltd.)

Although decades of research have shown associations between early caregiving adversity, stress physiology and limbic brain volume (e.g., amygdala, hippocampus), the developmental trajectories of these phenotypes are not well characterized. In the current study, we used an accelerated longitudinal design to assess the development of stress physiology, amygdala, and hippocampal volume following early institutional care. Previously Institutionalized (PI; N = 93) and comparison (COMP; N = 161) youth (ages 4-20 years old) completed 1-3 waves of data collection, each spaced approximately 2 years apart, for diurnal cortisol (N = 239) and structural MRI (N = 156). We observed a developmental shift in morning cortisol in the PI group, with blunted levels in childhood and heightened levels in late adolescence. PI history was associated with reduced hippocampal volume and reduced growth rate of the amygdala, resulting in smaller volumes by adolescence. Amygdala and hippocampal volumes were also prospectively associated with future morning cortisol in both groups. These results indicate that adversity-related physiological and neural phenotypes are not stationary during development but instead exhibit dynamic and interdependent changes from early childhood to early adulthood., (Copyright © 2021. Published by Elsevier Ltd.)

The quality of the early caregiving context sets the stage for the developing child's long term developmental trajectory. Infants are born highly dependent on parents and other caregivers for critical input for developing brain and behavioral systems. When infants experience early adversity, they are at risk for difficulties regulating behavior, emotions, and physiology. Parenting interventions have been developed to enhance parental responsiveness, thereby enhancing child outcomes. One such program, Attachment and Biobehavioral Catch-up (ABC), is a home visiting intervention designed to enhance parenting nurturance and sensitivity. In this paper, we will consider the importance of parental sensitivity and developmental consequences of sensitive and insensitive care. We will then describe interventions that target parental responsiveness and intervention effectiveness, focusing primarily on ABC. Public policy recommendations related to the importance of parental responsiveness will then be discussed., (Copyright © 2021 Elsevier Inc. All rights reserved.)

While antiretroviral therapy (ART) can completely suppress viremia, it is not a cure for HIV. HIV persists as a latent reservoir of infected cells, able to evade host immunity and re-seed infection following cessation of ART. Two promising immunotherapeutic strategies to eliminate both productively infected cells and reactivated cells of the reservoir are the adoptive transfer of potent HIV-specific T cells and the passive administration of HIV-specific broadly neutralizing antibodies also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). The simultaneous use of both as the basis of a single therapeutic has never been explored. We therefore sought to modify HIV-specific T cells from HIV-naive donors (to allow their use in the context of allotransplant, a promising platform for sterilizing cures) so they are able to secrete a broadly neutralizing antibody (bNAb) directed against the HIV envelope to elicit ADCC. We designed an antibody construct comprising bNAb 10-1074 heavy and light chains, fused to IgG3 Fc to elicit ADCC, with truncated cluster of differentiation 19 (CD19) as a selectable marker. HIV-specific T cells were expanded from HIV-naive donors by priming with antigen-presenting cells expressing overlapping HIV antigens in the presence of cytokines. T cells retained specificity against Gag, Nef, and Pol peptides (218.55 ± 300.14 interferon γ [IFNγ] spot-forming cells [SFC]/1 × 10 5 ) following transduction (38.92 ± 25.30) with the 10-1074 antibody constructs. These cells secreted 10-1074 antibodies (139.04 ± 114.42 ng/mL). The HIV-specific T cells maintained T cell function following transduction, and the secreted 10-1074 antibody bound HIV envelope (28.13% ± 19.42%) and displayed ADCC activity (10.47% ± 4.11%). Most critically, the 10-1074 antibody-secreting HIV-specific T cells displayed superior in vitro suppression of HIV replication. In summary, HIV-specific T cells can be engineered to produce antibodies mediating ADCC against HIV envelope-expressing cells. This combined innate/adaptive approach allows for synergy between the two immune arms, broadens the target range of the immune therapy, and provides further insight into what defines an effective anti-HIV response., (© 2020 The Authors.)

Curing HIV infection has been thwarted by the persistent reservoir of latently infected CD4 + T cells, which reinitiate systemic infection after antiretroviral therapy (ART) interruption. To evaluate reservoir depletion strategies, we developed a novel preclinical in vivo model consisting of immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells (PBMC) from long-term ART-suppressed HIV-infected donors. In the absence of ART, these mice developed rebound viremia which, 2 weeks after PBMC injection, was 1,000-fold higher (mean = 9,229,281 HIV copies/ml) in mice injected intrasplenically than in mice injected intraperitoneally (mean = 6,838 HIV copies/ml) or intravenously (mean = 591 HIV copies/ml). One week after intrasplenic PBMC injection, in situ hybridization of the spleen demonstrated extensive disseminated HIV infection, likely initiated from in vivo -reactivated primary latently infected cells. The time to viremia was delayed significantly by treatment with a broadly neutralizing antibody, 10-1074, compared to treatment with 10-1074-FcR null , suggesting that 10-1074 mobilized Fc-mediated effector mechanisms to deplete the replication-competent reservoir. This was supported by phylogenetic analysis of Env sequences from viral-outgrowth cultures and untreated, 10-1074-treated, or 10-1074-FcR null -treated mice. The predominant sequence cluster detected in viral-outgrowth cultures and untreated mouse plasma was significantly reduced in the plasma of 10-1074-treated mice, whereas two new clusters emerged that were not detected in viral-outgrowth cultures or plasma from untreated mice. These new clusters lacked mutations associated with 10-1074 resistance. Taken together, these data indicated that 10-1074 treatment depletes the reservoir of latently infected cells harboring replication competent HIV. Furthermore, this mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies. IMPORTANCE Sustained remission of HIV infection is prevented by a persistent reservoir of latently infected cells capable of reinitiating systemic infection and viremia. To evaluate strategies to reactivate and deplete this reservoir, we developed and characterized a new humanized mouse model consisting of highly immunodeficient mice intrasplenically injected with peripheral blood mononuclear cells from long-term ART-suppressed HIV-infected donors. Reactivation and dissemination of HIV infection was visualized in the mouse spleens in parallel with the onset of viremia. The applicability of this model for evaluating reservoir depletion treatments was demonstrated by establishing, through delayed time to viremia and phylogenetic analysis of plasma virus, that treatment of these humanized mice with a broadly neutralizing antibody, 10-1074, depleted the patient-derived population of latently infected cells. This mouse model represents a new in vivo approach for the preclinical evaluation of new HIV cure strategies., (Copyright © 2019 American Society for Microbiology.)

Efforts to cure human immunodeficiency virus (HIV) infection are obstructed by reservoirs of latently infected CD4 + T cells that can reestablish viremia. HIV-specific broadly neutralizing antibodies (bNAbs), defined by unusually wide neutralization breadths against globally diverse viruses, may contribute to the elimination of these reservoirs by binding to reactivated cells, thus targeting them for immune clearance. However, the relationship between neutralization of reservoir isolates and binding to corresponding infected primary CD4 + T cells has not been determined. Thus, the extent to which neutralization breadths and potencies can be used to infer the corresponding parameters of infected cell binding is currently unknown. We assessed the breadths and potencies of bNAbs against 36 viruses reactivated from peripheral blood CD4 + T cells from antiretroviral (ARV)-treated HIV-infected individuals by using paired neutralization and infected cell binding assays. Single-antibody breadths ranged from 0 to 64% for neutralization (80% inhibitory concentration [IC 80 ] of ≤10 μg/ml) and from 0 to 89% for binding, with two-antibody combinations (results for antibody combinations are theoretical/predicted) reaching levels of 0 to 83% and 50 to 100%, respectively. Infected cell binding correlated with virus neutralization for 10 of 14 antibodies (e.g., for 3BNC117, r  = 0.82 and P  < 0.0001). Heterogeneity was observed, however, with a lack of significant correlation for 2G12, CAP256.VRC26.25, 2F5, and 4E10. Our results provide guidance on the selection of bNAbs for interventional cure studies, both by providing a direct assessment of intra- and interindividual variabilities in neutralization and infected cell binding in a novel cohort and by defining the relationships between these parameters for a panel of bNAbs. IMPORTANCE Although antiretroviral therapies have improved the lives of people who are living with HIV, they do not cure infection. Efforts are being directed towards harnessing the immune system to eliminate the virus that persists, potentially resulting in virus-free remission without medication. HIV-specific antibodies hold promise for such therapies owing to their ability to both prevent the infection of new cells (neutralization) and direct the killing of infected cells. We isolated 36 HIV strains from individuals whose virus was suppressed by medication and tested 14 different antibodies for neutralization of these viruses and for binding to cells infected with the same viruses (critical for engaging natural killer cells). For both neutralization and infected cell binding, we observed variation both between individuals and amongst different viruses within an individual. For most antibodies, neutralization activity correlated with infected cell binding. These data provide guidance on the selection of antibodies for clinical trials., (Copyright © 2018 American Society for Microbiology.)

We previously isolated a herpes simplex virus 1 (HSV-1) mutant, KOS-NA, that carries two nonsynonymous mutations in UL39 , resulting in L393P and R950H amino acid substitutions in infected cell protein 6 (ICP6). Our published data studying KOS-NA pathogenesis strongly suggest that one of these ICP6 substitutions expressed from KOS-NA, R950H, severely impaired acute viral replication in the eyes and trigeminal ganglia of mice after inoculation onto the cornea and consequently impaired establishment and reactivation from latency. Because of its significant neuroattenuation, we tested KOS-NA as a potential prophylactic vaccine against HSV-1 in a mouse model of corneal infection. KOS-NA stimulated stronger antibody and T cell responses than a replication-competent ICP0-null mutant and a replication-incompetent ICP8-null mutant optimized for immunogenicity. Immunizations with the ICP0 - , ICP8 - , and KOS-NA viruses all reduced replication of wild-type HSV-1 challenge virus in the corneal epithelium to similar extents. Low immunizing doses of KOS-NA and the ICP8 - virus, but not the ICP0 - virus, protected mice against eyelid disease (blepharitis). Notably, only KOS-NA protected almost completely against corneal disease (keratitis) and greatly reduced latent infection by challenge virus. Thus, vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated disease of the eye, even at a very low immunizing dose. These results suggest that KOS-NA may be the foundation of an effective prophylactic vaccine to prevent or limit HSV-1 ocular diseases. IMPORTANCE HSV-1 is a ubiquitous human pathogen that infects the majority of the world's population. Although most infections are asymptomatic, HSV-1 establishes lifelong latency in infected sensory neurons, from which it can reactivate to cause deadly encephalitis or potentially blinding eye disease. No clinically effective vaccine is available. In this study, we tested the protective potential of a neuroattenuated HSV-1 mutant (KOS-NA) as a vaccine in mice. We compared the effects of immunization with KOS-NA to those of two other attenuated viruses, a replication-competent (ICP0 - ) virus and a replication-incompetent (ICP8 - ) virus. Our data show that KOS-NA proved superior to the ICP0- and ICP8-null mutants in protecting mice from corneal disease and latent infection. With its significant neuroattenuation, severe impairment in establishing latency, and excellent protective effect, KOS-NA represents a significant discovery in the field of HSV-1 vaccine development., (Copyright © 2018 American Society for Microbiology.)

Herpes simplex virus 1 (HSV-1) and HSV-2 are large, double-stranded DNA viruses that cause lifelong persistent infections characterized by periods of quiescence and recurrent disease. How HSV evolves within an infected individual experiencing multiple episodes of recurrent disease over time is not known. We determined the genome sequences of viruses isolated from two subjects in the Herpevac Trial for Women who experienced primary HSV-2 genital disease and compared them with sequences of viruses isolated from the subsequent fifth or sixth episode of recurrent disease in the same individuals. Each of the HSV-2 genome sequences was initially obtained using next-generation sequencing and completed with Sanger sequencing. Polymorphisms over the entire genomes were mapped, and amino acid variants resulting from nonsynonymous changes were analyzed based on the secondary and tertiary structures of a previously crystallized protein. A phylogenetic reconstruction was used to assess relationships among the four HSV-2 samples, other North American sequences, and reference sequences. Little genetic drift was detected in viruses shed by the same subjects following repeated reactivation events, suggesting strong selective pressure on the viral genome to maintain sequence fidelity during reactivations from its latent state within an individual host. Our results also demonstrate that some primary HSV-2 isolates from North America more closely resemble the HG52 laboratory strain from Scotland than the low-passage-number clinical isolate SD90e from South Africa or laboratory strain 333. Thus, one of the sequences reported here would be a logical choice as a reference strain for inclusion in future studies of North American HSV-2 isolates. IMPORTANCE The extent to which the HSV-2 genome evolves during multiple episodes of reactivation from its latent state within an infected individual is not known. We used next-generation sequencing techniques to determine whole-genome sequences of four viral samples from two subjects in the Herpevac Trial. The sequence of each subject's well-documented primary isolate was compared with the sequence of the isolate from their fifth or sixth episode of recurrent disease. Only 19 genetic polymorphisms unique to the primary or recurrent isolate were identified, 10 in subject A and 9 in subject B. These observations indicate remarkable genetic conservation between primary and recurrent episodes of HSV-2 infection and imply that strong selection pressures exist to maintain the fidelity of the viral genome during repeated reactivations from its latent state. The genome conservation observed also has implications for the potential success of a therapeutic vaccine., (Copyright © 2017 American Society for Microbiology.)

The Herpevac Trial for Women revealed that three dose HSV-2 gD vaccine was 58% protective against culture-positive HSV-1 genital disease, but it was not protective against HSV-2 infection or disease. To determine whether vaccine-induced immune responses had selected for a particular gD sequence in strains infecting vaccine recipients compared with viruses infecting control subjects, genetic sequencing studies were carried out on viruses isolated from subjects infected with HSV-1 or HSV-2. We identified naturally occurring variants among the gD sequences obtained from 83 infected subjects. Unique or low frequency amino acid substitutions in the ectodomain of gD were found in 6 of 39 HSV-1-infected subjects and in 7 of 44 HSV-2-infected subjects. However, no consistent amino acid change was identified in isolates from gD-2 vaccine recipients compared with infected placebo recipients. gC and gE surround and partially shield gD from neutralizing antibody, and gB also participates closely in the viral entry process. Therefore, these genes were sequenced from a number of isolates to assess whether sequence variation may alter protein conformation and influence the virus strain's capacity to be neutralized by vaccine-induced antibody. gC and gE genes sequenced from HSV-1-infected subjects showed more variability than their HSV-2 counterparts. The gB sequences of HSV-1 oral isolates resembled each other more than they did gB sequences rom genital isolates. Overall, however, comparison of glycoprotein sequences of viral isolates obtained from infected subjects did not reveal any singular selective pressure on the viral cell attachment protein or surrounding glycoproteins due to administration of gD-2 vaccine.

Orthopoxviruses continue to pose a significant threat to the population as potential agents of bioterrorism. An intentional release of natural or engineered variola virus (VARV) or monkeypox viruses would cause mortality and morbidity in the target population. To address this, antivirals have been developed and evaluated in animal models of smallpox and monkeypox. One such antiviral, brincidofovir (BCV, previously CMX001), has demonstrated high levels of efficacy against orthopoxviruses in animal models and is currently under clinical evaluation for prevention and treatment of diseases caused by cytomegaloviruses and adenoviruses. In this study we use the mousepox model of smallpox to evaluate the relationship between the magnitude of the infectious virus dose and an efficacious BCV therapy outcome when treatment is initiated concomitant with detection of ectromelia virus viral DNA (vDNA) in mouse buccal swabs. We found that vDNA could be detected in buccal swabs of some, but not all infected mice over a range of challenge doses by day 3 or 4 postexposure, when initiation of BCV treatment was efficacious, suggesting that detection of vDNA in buccal swabs could be used as a trigger to initiate BCV treatment of an entire potentially exposed population. However, buccal swabs of some mice did not become positive until 5 days postexposure, when initiation of BCV therapy failed to protect mice that received high doses of virus. And finally, the data suggest that the therapeutic window for efficacious BCV treatment decreases as the virus infectious dose increases. Extrapolating these findings to VARV, the data suggest that treatment should be initiated as soon as possible after exposure and not rely on a diagnostic tool such as the measurement of vDNA in buccal cavity swabs; however, consideration should be given to the fact that the behavior/disease-course of VARV in humans is different from that of ectromelia virus in the mouse., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Herpes simplex virus 1 (HSV-1) and HSV-2 remain major human pathogens despite the development of anti-HSV therapeutics as some of the first antiviral drugs. Current therapies are incompletely effective and frequently drive the evolution of drug-resistant mutants. We recently determined that certain natural troponoid compounds such as β-thujaplicinol readily suppress HSV-1 and HSV-2 replication. Here, we screened 26 synthetic α-hydroxytropolones with the goals of determining a preliminary structure-activity relationship for the α-hydroxytropolone pharmacophore and providing a starting point for future optimization studies. Twenty-five compounds inhibited HSV-1 and HSV-2 replication at 50 μM, and 10 compounds inhibited HSV-1 and HSV-2 at 5 μM, with similar inhibition patterns and potencies against both viruses being observed. The two most powerful inhibitors shared a common biphenyl side chain, were capable of inhibiting HSV-1 and HSV-2 with a 50% effective concentration (EC50) of 81 to 210 nM, and also strongly inhibited acyclovir-resistant mutants. Moderate to low cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50] of 50 to >100 μM). Therapeutic indexes ranged from >170 to >1,200. These data indicate that troponoids and specifically α-hydroxytropolones are a promising lead scaffold for development as anti-HSV drugs provided that toxicity can be further minimized. Troponoid drugs are envisioned to be employed alone or in combination with existing nucleos(t)ide analogs to suppress HSV replication far enough to prevent viral shedding and to limit the development of or treat nucleos(t)ide analog-resistant mutants., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency.

Herpesviruses are large double-stranded DNA viruses that cause serious human diseases. Herpesvirus DNA replication depends on multiple processes typically catalyzed by nucleotidyltransferase superfamily (NTS) enzymes. Therefore, we investigated whether inhibitors of NTS enzymes would suppress replication of herpes simplex virus 1 (HSV-1) and HSV-2. Eight of 42 NTS inhibitors suppressed HSV-1 and/or HSV-2 replication by >10-fold at 5 μM, with suppression at 50 μM reaching ∼1 million-fold. Five compounds in two chemical families inhibited HSV replication in Vero and human foreskin fibroblast cells as well as the approved drug acyclovir did. The compounds had 50% effective concentration values as low as 0.22 μM with negligible cytotoxicity in the assays employed. The inhibitors suppressed accumulation of viral genomes and infectious particles and blocked events in the viral replication cycle before and during viral DNA replication. Acyclovir-resistant mutants of HSV-1 and HSV-2 remained highly sensitive to the NTS inhibitors. Five of six NTS inhibitors of the HSVs also blocked replication of another herpesvirus pathogen, human cytomegalovirus. Therefore, NTS enzyme inhibitors are promising candidates for new herpesvirus treatments that may have broad efficacy against members of the herpesvirus family., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The γ34.5 gene of herpes simplex virus (HSV) 2 encodes ICP34.5, which enhances HSV-2 neurovirulence by an unknown mechanism. We found that an HSV-2 γ34.5-null mutant (γ34.5(-/-)) replicated less robustly than its rescue virus (γ34.5R) in wild-type mouse embryo fibroblasts (MEFs), and in cells primed with IFNβ. Increased eIF2α phosphorylation correlated with γ34.5(-/-) attenuation. However, γ34.5(-/-) achieved titers equivalent to γ34.5R in MEFs lacking the type I IFN receptor (IFNα/βR(-/-)) or lacking protein kinase R. γ34.5(-/-) also replicated poorly in the vaginal mucosa of wild-type mice, caused little genital inflammation, and spread to the nervous system at lower levels compared to γ34.5R. In IFNα/βR(-/-) mice, however, γ34.5(-/-) regained the capacity to replicate and cause disease equivalent to γ34.5R after intravaginal infection or direct inoculation into the central nervous system. Thus, the capacity of HSV-2 ICP34.5 to interdict the type I IFN response in vivo largely determines its neurovirulence., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Unlabelled: The herpes simplex virus 1 (HSV-1) ICP34.5 protein strongly influences neurovirulence and regulates several cellular antiviral responses. Despite the clinical importance of HSV-2, relatively little is known about its ICP34.5 ortholog. We found that HSV-2 produces up to four distinct forms of ICP34.5 in infected cells: a full-length protein, one shorter form sharing the N terminus, and two shorter forms sharing the C terminus. These forms appeared with similar kinetics and accumulated in cells over much of the replication cycle. We confirmed that the N-terminal form is translated from the primary unspliced transcript to a stop codon within the intron unique to HSV-2 γ34.5. We found that the N-terminal form was produced in a variety of cell types and by 9 of 10 clinical isolates. ICP27 influenced but was not required for expression of the N-terminal form. Western blotting and reverse transcription-PCR indicated the C-terminal forms did not contain the N terminus and were not products of alternative splicing or internal transcript initiation. Expression plasmids encoding methionine at amino acids 56 and 70 generated products that comigrated in SDS-PAGE with the C1 and C2 forms, respectively, and mutation of these sites abolished C1 and C2. Using a recombinant HSV-2 encoding hemagglutinin (HA)-tagged ICP34.5, we demonstrated that the C-terminal forms were also produced during infection of many human and mouse cell types but were not detectable in mouse primary neurons. The protein diversity generated from the HSV-2 γ34.5 open reading frame implies additional layers of cellular regulation through potential independent activities associated with the various forms of ICP34.5., Importance: The herpes simplex virus 1 (HSV-1) protein ICP34.5, encoded by the γ34.5 gene, interferes with several host defense mechanisms by binding cellular proteins that would otherwise stimulate the cell's autophagic, translational-arrest, and type I interferon responses to virus infection. ICP34.5 also plays a crucial role in determining the severity of nervous system infections with HSV-1 and HSV-2. The HSV-2 γ34.5 gene contains an intron not present in HSV-1 γ34.5. A shorter N-terminal form of HSV-2 ICP34.5 can be translated from the unspliced γ34.5 mRNA. Here, we show that two additional forms consisting of the C-terminal portion of ICP34.5 are generated in infected cells. Production of these N- and C-terminal forms is highly conserved among HSV-2 strains, including many clinical isolates, and they are broadly expressed in several cell types, but not mouse primary neurons. Multiple ICP34.5 polypeptides add additional complexity to potential functional interactions influencing HSV-2 neurovirulence., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The cellular prion protein (PrP) often plays a cytoprotective role by regulating autophagy in response to cell stress. The stress of infection with intracellular pathogens can stimulate autophagy, and autophagic degradation of pathogens can reduce their replication and thus help protect the infected cells. PrP also restricts replication of several viruses, but whether this activity is related to an effect on autophagy is not known. Herpes simplex virus 1 (HSV-1) effectively counteracts autophagy through binding of its ICP34.5 protein to the cellular proautophagy protein beclin-1. Autophagy can reduce replication of an HSV-1 mutant, Δ68H, which is incapable of binding beclin-1. We found that deletion of PrP in mice complements the attenuation of Δ68H, restoring its capacity to replicate in the central nervous system (CNS) to wild-type virus levels after intracranial or corneal infection. Cultured primary astrocytes but not neurons derived from PrP(-/-) mice also complemented the attenuation of Δ68H, enabling Δ68H to replicate at levels equivalent to wild-type virus. Ultrastructural analysis showed that normal astrocytes exhibited an increase in the number of autophagosomes after infection with Δ68H compared with wild-type virus, but PrP(-/-) astrocytes failed to induce autophagy in response to Δ68H infection. Redistribution of EGFP-LC3 into punctae occurred more frequently in normal astrocytes infected with Δ68H than with wild-type virus, but not in PrP(-/-) astrocytes, corroborating the ultrastructural analysis results. Our results demonstrate that PrP is critical for inducing autophagy in astrocytes in response to HSV-1 infection and suggest that PrP positively regulates autophagy in the mouse CNS.

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) has endoribonuclease activity and rapidly reduces protein synthesis in infected cells through mRNA degradation. Herpes simplex virus 1 (HSV-1) and HSV-2 vhs mutants are highly attenuated in vivo, but replication and virulence are largely restored to HSV-2 vhs mutants in the absence of a type I interferon (IFN) response. The role of vhs in pathogenesis and the hindrance of the type I IFN response have classically been examined with viruses that completely lack vhs or express a truncated vhs protein. To determine whether RNase activity is the principal mechanism of vhs-mediated type I IFN resistance and virulence, we constructed a HSV-2 point mutant that synthesizes full-length vhs protein lacking RNase activity (RNase(-) virus). Wild-type and mutant HSV-2 vhs proteins coimmunoprecipitated with VP16 and VP22. vhs protein bearing the point mutation was packaged into the virion as efficiently as the wild-type vhs protein. Like a mutant encoding truncated vhs, the RNase(-) virus showed IFN-dependent replication that was restricted compared with that of the wild-type virus. The RNase(-) virus was highly attenuated in wild-type mice infected intravaginally, with reduced mucosal replication, disease severity, and spread to the nervous system comparable to those of the vhs truncation mutant. Surprisingly, in alpha/beta interferon (IFN-alpha/beta) receptor knockout mice, the vhs RNase mutant was more attenuated than the vhs truncation mutant in terms of disease severity and virus titer in vaginal swabs and central nervous system samples, suggesting that non-enzymatically active vhs protein interferes with efficient virus replication. Our results indicate that vhs enzymatic activity plays a complex role in vhs-mediated type I IFN resistance during HSV-2 infection.

The Drosophila melanogaster Nipped-B protein facilitates transcriptional activation of the cut and Ultrabithorax genes by remote enhancers. Sequence homologues of Nipped-B, Scc2 of Saccharomyces cerevisiae, and Mis4 of Schizosaccharomyces pombe are required for sister chromatid cohesion during mitosis. The evolutionarily conserved Cohesin protein complex mediates sister chromatid cohesion, and Scc2 and Mis4 are needed for Cohesin to associate with chromosomes. Here, we show that Nipped-B is also required for sister chromatid cohesion but that, opposite to the effect of Nipped-B, the stromalin/Scc3 component of Cohesin inhibits long-range activation of cut. To explain these findings, we propose a model based on the chromatin domain boundary activities of Cohesin in which Nipped-B facilitates cut activation by alleviating Cohesin-mediated blocking of enhancer-promoter communication.

Sex dependence of copper, zinc, iron nutritional status and hepatic lipid peroxidation was studied in gonadectomized, hormone substituted and sham-operated male and female rats. The experiment was performed on H-Wistar rats (106 +/- 10 g) fed conventional laboratory food ad libitum for 12 weeks. The estrogen dependence of copper status is fully confirmed by the results of this study. In hormonally active females the malondialdehyde production was depressed which is supposed to be connected with high copper and low triglyceride levels in the liver. The anaemia observed in estrogen substituted rats may be a result of lower lipid peroxidation rate. The levels of retinol and alkaline phosphatase activity in sera are probably influenced by estrogen action. It is suggested that testosterone leads to acceleration of lipid peroxidation.

Factors influencing the effectivity of replacement therapy with Panpur and Creon were controlled by in vivo and in vitro investigations. Both enteric coated preparations were equally acid protected, they even seemed to be more effective in hyperacid than in anacid chronic pancreatitis patients. Thus the uneven results of Panpur treatment in pancreatic steatorrhea cannot be explained by acid inactivation of the enzymes. Creon dose-dependently ameliorated the steatorrhea as well as vitamin B12 absorption while crushed but not the intact Panpur has only some insignificant effect. Good mixing of pancreatin with the B12-intrinsic factor - R protein complex and with the protein containing meal seems to be important for digestion of protein as well as fat. Unbound, overflowing trypsin activity of Panpur resulted in fast proteolytic inactivation of lipase. This could be diminished by soybean trypsin inhibitor which increased the in vivo effectiveness of the preparate. In summary Creon fulfilled two important factors of replacement therapy more successfully than Panpur: good mixing with meals and stability of lipase against proteolytic splitting, that is why it proved to be more effective for replacement therapy of pancreatic insufficiency.