The details of the pediatric immune system that supports induction of antibodies capable of neutralizing geographically-diverse or heterologous HIV-1 is currently unclear. Here we explore the pediatric immune environment in neonatal macaque undergoing Simian-HIV infection. Simian-HIV infection of 11 pairs of therapy-naive dams and infant rhesus macaques for 24 months results in heterologous HIV-1 neutralizing antibodies in 64% of young macaques compared to 18% of adult macaques. Heterologous HIV-1 neutralizing antibodies emerge by 12 months post-infection in young macaques, in association with lower expression of immunosuppressive genes, fewer germinal center CD4 + T regulatory cells, and a lower ratio of CD4 + T follicular regulatory to helper cells. Antibodies from peripheral blood B cells in two young macaques following SHIV infection neutralize 13% of 119 heterologous HIV-1 strains and map to regions of canonical broadly neutralizing antibody epitopes on the envelope surface protein. Here we show that pediatric immunity to SHIV infection in a macaque model may inform vaccine strategies to induce effective HIV-1 neutralizing antibodies in infants and children prior to viral exposure. The specifics of the pediatric immune response that gives rise to antibodies capable of neutralising diverse HIV-1 strains is not fully understood. Here the authors characterise the immune environment of Simian-HIV infected paediatric macaques and link to antibody neutralisation induction.

Kaposi's sarcoma-associated herpesvirus (KSHV)-associated primary effusion lymphomas (PEL) are traditionally viewed as homogenous regarding viral transcription and lineage of origin, but so far this contention has not been explored at the single-cell level. Single-cell RNA sequencing of latently infected PEL supports the existence of multiple subpopulations even within a single cell line. At most 1% of the cells showed evidence of near-complete lytic transcription. The majority of cells only expressed the canonical viral latent transcripts: those originating from the latency locus, the viral interferon regulatory factor locus, and the viral lncRNA nut-1/Pan/T1.1; however, a significant fraction of cells showed various degrees of more permissive transcription, and some showed no evidence of KSHV transcripts whatsoever. Levels of viral interleukin-6 (IL-6)/K2 mRNA emerged as the most distinguishing feature to subset KSHV-infected PEL. One newly uncovered phenotype is the existence of BCBL-1 cells that readily adhered to fibronectin and that displayed mesenchymal lineage-like characteristics. IMPORTANCE Latency is the defining characteristic of the Herpesviridae and central to the tumorigenesis phenotype of Kaposi's sarcoma-associated herpesvirus (KSHV). KSHV-driven primary effusion lymphomas (PEL) rapidly develop resistance to therapy, suggesting tumor instability and plasticity. At any given time, a fraction of PEL cells spontaneously reactivate KSHV, suggesting transcriptional heterogeneity even within a clonal cell line under optimal growth conditions. This study employed single-cell mRNA sequencing to explore the within-population variability of KSHV transcription and how it relates to host cell transcription. Individual clonal PEL cells exhibited differing patterns of viral transcription. Most cells showed the canonical pattern of KSHV latency (LANA, vCyc, vFLIP, Kaposin, and vIRFs), but a significant fraction evidenced extended viral gene transcription, including of the viral IL-6 homolog, open reading frame K2. This study suggests new targets of intervention for PEL. It establishes a conceptual framework to design KSHV cure studies analogous to those for HIV.

The majority of new HIV-1 infections occur in young adults, with adolescent women being 3 times more likely to acquire HIV than young men. Implementation of HIV prevention strategies has been less successful in this age group; thus, a vaccine given prior to adolescence remains a high priority. We propose that instead of starting HIV vaccination during adolescence, an HIV vaccine regimen initiated in early infancy, aligned with the well-accepted pediatric vaccine schedule and followed with booster immunizations, will provide an alternative means to reduce HIV acquisition in adolescence. Importantly, the long window of time between the first infant vaccine dose and the adolescence vaccine dose will allow for the maturation of highly functional HIV Env-specific antibody responses. Our study provides evidence that early life vaccination induces durable Env-specific plasma IgG responses that can be boosted to further improve the quality of the antibody response.

Nonhuman primate (NHP) models are invaluable for HIV pathogenesis, intervention and cure studies. To enhance the translational potential of NHP HIV vaccine studies, clinically relevant R5-tropic, tier 2 neutralization sensitive, and mucosally transmissible simian-human immunodeficiency viruses (SHIVs) have been designed. Towards our goal of developing vaccines to prevent breastmilk transmission of HIV, we evaluated virological outcomes of three distinct SHIVs in repeated weekly oral exposure regimens in infant rhesus macaques. The selected strains SHIV-1157ipd3N4, SHIV-1157(QNE)Y173H, and SHIV CH505 375H.dCT express a clade C HIV Env, the clade most prevalent in regions with high pediatric HIV infections.All three SHIVs were orally transmissible. However, compared to the pediatric SIVmac251 model, SHIV replication was more attenuated. Some animals exposed to weekly low-dose (20 TCID50) SHIV-1157ipd3N4 had transient viremia blips associated with lack or delayed seroconversion. Animals with acute viremia ≥10,000 viral RNA copies/ml seroconverted. This finding was reminiscent of an earlier study suggesting to continue challenges until a threshold of ≥10,000 viral RNA copies/ml is surpassed to achieve seroconversion, one criterion of HIV diagnosis. All animals exposed weekly with higher doses (>104 TCID50) of SHIV-1157(QNE)Y173H or SHIV CH505 375h.dCT developed persistent infection with high peak viremia and seroconverted. Chronic viremia varied widely in all three SHIV infection models. Thus, although R5 clade C SHIVs are excellent tools to study prevention of virus acquisition, secondary virological outcomes of vaccine efficacy (e.g. risk of infection per exposure, modulation of peak viremia, viral set point) will require careful consideration and validation in SHIV challenge models.IMPORTANCE The development of an effective HIV vaccine remains a top priority towards the goal of reducing the number of new HIV infections. Studies in nonhuman primate models of HIV infection have been instrumental in the preclinical evaluation of candidate vaccines. To assess the role of HIV Env-specific antibodies with broadly neutralizing or Fc-mediated effector function in these NHP models, the development and optimization of novel SHIV challenge models is required. We evaluate three different clade C HIV Env SHIVs for infectivity of infant macaques by the oral route. Our results demonstrate that SHIV-1157ipd3N4, SHIV-1157(QNE)Y173H, and SHIV CH505 375H.dCT can be orally transmitted and establish persistent infection in infant rhesus macaques, but chronic viremia levels vary widely. Therefore, SHIV infection models are most pertinent when prevention of systemic infection is the primary readout of vaccine efficacy, but secondary outcome measures of vaccine efficacy will require stringent criteria and extensive validation.

In 2014, an estimated 220,000 children became newly HIV-infected, with 50% being transmitted via breast-feeding. The present study assessed whether inclusion of an oral route of immunization would increase pediatric vaccine efficacy in the rhesus macaque model of oral SIV infection. Two groups of 6 neonatal macaques received 2 DNA-SIV immunizations at weeks 0 and 3, followed by 2 boosts with modified vaccinia Ankara virus (MVA)-SIVgag/pol/env at weeks 6 and 9. Group A was immunized by the intramuscular (IM) route. Infants in Group B received the DNA-prime both per-orally (PO) and IM and MVA-SIV was administered both by the sublingual (SL) and IM route. Mock control animals received saline immunizations PO+IM at weeks 0 and 3 and mock MVA vector SL+IM at weeks 6 and 9. At week 12, all infants were challenged once weekly using a low-dose (102 TCID50) oral SIVmac251 challenge regimen until systemic infection was confirmed by PCR. Statistical significance was determined using nonparametric Mann-Whitney or Kruskal-Wallis tests with p<0.05. Infants vaccinated by the SL/IM route had a significantly lower per-exposure risk of oral SIV infection (p<0.0001; log-ranked Mantel-Cox test) compared to mock- and IM/IM-vaccinated animals and lower peak viremia (p=0.03) compared to mock controls. Control of viremia was associated with higher fecal IgG responses to SIV V1V2 (p=0.0087) and p55 gag (p=0.0087) at the time of oral challenge initiation. In contrast, plasma SIV-specific antibodies did not influence challenge outcome. These data indicate that the inclusion of an oral route in a pediatric vaccine to prevent oral SIV infection is superior to an IM only regimen and emphasizes the importance of enhancing mucosal immune responses via local immunization.

Most infant deaths occur in the first year of life. Yet, our knowledge of immune development during this period is scarce and derived from cord blood (CB) only. To more effectively combat pediatric diseases, a deeper understanding of the kinetics and the factors that regulate the maturation of immune functions in early life is needed. Increased disease susceptibility of infants is generally attributed to T helper 2-biased immune responses. The differentiation of CD4+ T cells along a specific T helper cell lineage is dependent on the pathogen type, and on costimulatory and cytokine signals provided by antigen-presenting cells. Cytokines also regulate many other aspects of the host immune response. Therefore, toward the goal of increasing our knowledge of early immune development, we defined the temporal development of the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling function of CD4+ T cells using cross-sectional blood samples from healthy infants ages 0 (birth) to 14 months. We specifically focused on cytokines important in T cell differentiation (IFN-γ, IL-12, and IL-4) or in T cell survival and expansion (IL-2 and IL-7) in infant CD4+ T cells. Independent of the cytokine tested, JAK/STAT signaling in infant compared to adult CD4+ T cells was impaired at birth, but increased during the first year, with the most pronounced changes occurring in the first 6 months. The relative change in JAK/STAT signaling of infant CD4+ T cells with age was distinct for each cytokine tested. Thus, while about 60% of CB CD4+ T cells could efficiently activate STAT6 in response to IL-4, less than 5% of CB CD4+ T cells were able to activate the JAK/STAT pathway in response to IFN-γ, IL-12 or IL-2. By 4-6 months of age, the activation of the cytokine-specific STAT molecules was comparable to adults in response to IL-4 and IFN-γ, while IL-2- and IL-12-induced STAT activation remained below adult levels even at 1 year. These results suggest that common developmental and cytokine-specific factors regulate the maturation of the JAK/STAT signaling function in CD4+ T cells during the first year of life.

Abstract Natural Killer (NK) cells are an important component of the innate immune system, capable of providing a fast and effective response against virally infected cells. NK cells are mainly characterized by their cytotoxic function and their ability to secrete cytokines. It has been shown that infant NK cells have decreased cytotoxicity and cytokine-secreting function, suggesting hyporesponsiveness of NK cells during the first year of life. Because NK cell activation is dependent on cytokine stimulation, we hypothesized that infant NK cells are hyporesponsive due to deficiencies in signaling following cytokine stimulation. We examined the activation of human infant and adult NK cells in response to IL2, IL12 or IFNα stimulation, cytokines important in NK survival and function. Using Phosflow analysis, we measured the phosphorylation of transcription factors, STATs, specific for the distinct cytokines. Cord blood NK cells showed significantly lower pSTAT activation when compared to adult NK cells when treated with IL2 or IFNα but not IL12. However, despite pSTAT4 activation in response to IL12, no nuclear translocation was observed by ImageStreamX Mark II analysis. NK cells in 6 and 12-month-old infants showed similar frequencies of pSTAT NK cells compared to adults when treated with IL2, IL12 or IFNα, suggesting that cytokine signaling in NK cells is age-dependent. Surprisingly, pSTAT activation was restored in cord blood NK cells when treated with a cocktail of IL2, IL12 and IL15. These results suggest that JAK/STAT signaling in infant NK cells is impaired at different steps in the pathway in response to specific cytokines.

Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansions of the (ATTCT)n repeat within an intron of the human ATXN10 gene. In contrast to other expandable repeats, this pentanucleotide repeat does not form stable intra- or interstranded DNA structures, being a DNA unwinding element instead. We analyzed the instability of the (ATTCT)n repeat in a yeast experimental system, where its expansions led to inactivation of the URA3 reporter gene. The inactivation was due to a dramatic decrease in the mRNA levels owing to premature transcription termination and RNA polyadenylation at the repeat. The rates of expansions strongly increased with the repeat's length, mimicking genetic anticipation in human pedigrees. A first round of genetic analysis showed that a functional TOF1 gene precludes, whereas a functional RAD5 gene promotes, expansions of the (ATTCT)n repeat. We hypothesize that repeat expansions could occur upon fortuitous template switching during DNA replication. The rate of repeat contractions was elevated in the Tof1 knockout strain, but it was not affected by the RAD5 gene. Supporting the notion of replication irregularities, we found that (ATTCT)n repeats also cause length-dependent chromosomal fragility in yeast. Repeat-mediated fragility was also affected by the Tof1 and Rad5 proteins, being reduced in their absence.