Microbial Translocation and Inflammation Occur in Hyperacute Immunodeficiency Virus Infection and Compromise Host Control of Virus Replication.

Within the first three weeks of human immunodeficiency virus (HIV) infection, virus replication peaks in peripheral blood. Despite the critical, causal role of virus replication in determining transmissibility and kinetics of progression to acquired immune deficiency syndrome (AIDS), there is limited understanding of the conditions required to transform the small localized transmitted founder virus population into a large and heterogeneous systemic infection. Here we show that during the hyperacute "pre-peak" phase of simian immunodeficiency virus (SIV) infection in macaques, high levels of microbial DNA transiently translocate into peripheral blood. This, heretofore unappreciated, hyperacute-phase microbial translocation was accompanied by sustained reduction of lipopolysaccharide (LPS)-specific antibody titer, intestinal permeability, increased abundance of CD4+CCR5+ T cell targets of virus replication, and T cell activation. To test whether increasing gastrointestinal permeability to cause microbial translocation would amplify viremia, we treated two SIV-infected macaque 'elite controllers' with a short-course of dextran sulfate sodium (DSS)-stimulating a transient increase in microbial translocation and a prolonged recrudescent viremia. Altogether, our data implicates translocating microbes as amplifiers of immunodeficiency virus replication that effectively undermine the host's capacity to contain infection.

Deep Sequencing Reveals Potential Antigenic Variants at Low Frequencies in Influenza A Virus-Infected Humans.

UNLABELLED: Influenza vaccines must be frequently reformulated to account for antigenic changes in the viral envelope protein, hemagglutinin (HA). The rapid evolution of influenza virus under immune pressure is likely enhanced by the virus's genetic diversity within a host, although antigenic change has rarely been investigated on the level of individual infected humans. We used deep sequencing to characterize the between- and within-host genetic diversity of influenza viruses in a cohort of patients that included individuals who were vaccinated and then infected in the same season. We characterized influenza HA segments from the predominant circulating influenza A subtypes during the 2012-2013 (H3N2) and 2013-2014 (pandemic H1N1; H1N1pdm) flu seasons. We found that HA consensus sequences were similar in nonvaccinated and vaccinated subjects. In both groups, purifying selection was the dominant force shaping HA genetic diversity. Interestingly, viruses from multiple individuals harbored low-frequency mutations encoding amino acid substitutions in HA antigenic sites at or near the receptor-binding domain. These mutations included two substitutions in H1N1pdm viruses, G158K and N159K, which were recently found to confer escape from virus-specific antibodies. These findings raise the possibility that influenza antigenic diversity can be generated within individual human hosts but may not become fixed in the viral population even when they would be expected to have a strong fitness advantage. Understanding constraints on influenza antigenic evolution within individual hosts may elucidate potential future pathways of antigenic evolution at the population level. IMPORTANCE: Influenza vaccines must be frequently reformulated due to the virus's rapid evolution rate. We know that influenza viruses exist within each infected host as a "swarm" of genetically distinct viruses, but the role of this within-host diversity in the antigenic evolution of influenza has been unclear. We characterized here the genetic and potential antigenic diversity of influenza viruses infecting humans, some of whom became infected despite recent vaccination. Influenza virus between- and within-host genetic diversity was not significantly different in nonvaccinated and vaccinated humans, suggesting that vaccine-induced immunity does not exert strong selective pressure on viruses replicating in individual people. We found low-frequency mutations, below the detection threshold of traditional surveillance methods, in nonvaccinated and vaccinated humans that were recently associated with antibody escape. Interestingly, these potential antigenic variants did not reach fixation in infected people, suggesting that other evolutionary factors may be hindering their emergence in individual humans.

Antibody-dependent cell-mediated cytotoxicity antibody responses to inactivated and live-attenuated influenza vaccination in children during 2014-15.

BACKGROUND: Seasonal influenza vaccines aim to induce strain-specific neutralizing antibodies. Non-neutralizing antibodies may be more broadly cross-reactive and still protect through mechanisms including antibody-dependent cell-mediated cytotoxicity (ADCC). Influenza vaccines may stimulate ADCC antibodies in adults, but whether they do so in children is unknown. Here we examined how vaccination affects cross-reactive ADCC antibody responses in children after receipt of inactivated trivalent vaccine (IIV3) or quadrivalent live-attenuated vaccine (LAIV4). METHODS: Children aged 5-17 were recruited in fall 2014 to provide pre- and post-vaccination serum samples. Children aged 5-9 received LAIV4 based on then-current recommendation, and older children were randomly assigned to IIV3 or LAIV4. We used microtiter-plate-based flow cytometry with an NK cell line to examine ADCC antibody responses to the 2014-15 H3N2 vaccine component (A/Texas/50/2012 [TX12]) and a drifted strain, A/Switzerland/9715293/2013 (SW13). Responses were stratified by two-season (2013-14 and 2014-15) vaccine sequence. RESULTS: Eighty-five children received LAIV4 and 45 received IIV3. Prevaccination ADCC activity was highest in children who had received any vaccine in the prior season. Increase in ADCC antibody responses against the vaccine strain TX12 following vaccination was greatest for participants who received IIV3 in 2014-15 and LAIV4 in the prior season (geometric mean fold rise [MFR] = 1.6, 95% CI. 1.23-2.11). This group also had a detectable ADCC response to the drifted SW13 strain. There was a modest ADCC response against SW13 in LAIV4 recipients who were unvaccinated in the previous season (MFR = 1.18, 95% CI 1.10-1.25). There were no significant changes in 2014-15 ADCC response to vaccination among children who had received IIV3 in 2013-14. CONCLUSIONS: Vaccinating children with IIV3 after prior receipt of LAIV4 generated a modest increase in ADCC antibodies, including some cross-reactivity with an emerging drift variant. Other vaccine-induced ADCC responses were minimal and not affected by vaccine type or sequence.

Gut microbiomes of mobile predators vary with landscape context and species identity.

Landscape context affects predator-prey interactions and predator diet composition, yet little is known about landscape effects on insect gut microbiomes, a determinant of physiology and condition. Here, we combine laboratory and field experiments to examine the effects of landscape context on the gut bacterial community and body condition of predatory insects. Under laboratory conditions, we found that prey diversity increased bacterial richness in insect guts. In the field, we studied the performance and gut microbiota of six predatory insect species along a landscape complexity gradient in two local habitat types (soybean fields vs. prairie). Insects from soy fields had richer gut bacteria and lower fat content than those from prairies, suggesting better feeding conditions in prairies. Species origin mediated landscape context effects, suggesting differences in foraging of exotic and native predators on a landscape scale. Overall, our study highlights complex interactions among gut microbiota, predator identity, and landscape context.

A modified vaccinia Ankara vaccine vector expressing a mosaic H5 hemagglutinin reduces viral shedding in rhesus macaques.

The rapid antigenic evolution of influenza viruses requires frequent vaccine reformulations. Due to the economic burden of continuous vaccine reformulation and the threat of new pandemics, there is intense interest in developing vaccines capable of eliciting broadly cross-reactive immunity to influenza viruses. We recently constructed a "mosaic" hemagglutinin (HA) based on subtype 5 HA (H5) and designed to stimulate cellular and humoral immunity to multiple influenza virus subtypes. Modified vaccinia Ankara (MVA) expressing this H5 mosaic (MVA-H5M) protected mice against multiple homosubtypic H5N1 strains and a heterosubtypic H1N1 virus. To assess its potential as a human vaccine we evaluated the ability of MVA-H5M to provide heterosubtypic immunity to influenza viruses in a non-human primate model. Rhesus macaques received an initial dose of either MVA-H5M or plasmid DNA encoding H5M, followed by a boost of MVA-H5M, and then were challenged, together with naïve controls, with the heterosubtypic virus A/California/04/2009 (H1N1pdm). Macaques receiving either vaccine regimen cleared H1N1pdm challenge faster than naïve controls. Vaccination with H5M elicited antibodies that bound H1N1pdm HA, but did not neutralize the H1N1pdm challenge virus. Plasma from vaccinated macaques activated NK cells in the presence of H1N1pdm HA, suggesting that vaccination elicited cross-reactive antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Although HA-specific T cell responses to the MVA-H5M vaccine were weak, responses after challenge were stronger in vaccinated macaques than in control animals. Together these data suggest that mosaic HA antigens may provide a means for inducing broadly cross-reactive immunity to influenza viruses.

Selective Bottlenecks Shape Evolutionary Pathways Taken during Mammalian Adaptation of a 1918-like Avian Influenza Virus.

Avian influenza virus reassortants resembling the 1918 human pandemic virus can become transmissible among mammals by acquiring mutations in hemagglutinin (HA) and polymerase. Using the ferret model, we trace the evolutionary pathway by which an avian-like virus evolves the capacity for mammalian replication and airborne transmission. During initial infection, within-host HA diversity increased drastically. Then, airborne transmission fixed two polymerase mutations that do not confer a detectable replication advantage. In later transmissions, selection fixed advantageous HA1 variants. Transmission initially involved a "loose" bottleneck, which became strongly selective after additional HA mutations emerged. The stringency and evolutionary forces governing between-host bottlenecks may therefore change throughout host adaptation. Mutations occurred in multiple combinations in transmitted viruses, suggesting that mammalian transmissibility can evolve through multiple genetic pathways despite phenotypic constraints. Our data provide a glimpse into avian influenza virus adaptation in mammals, with broad implications for surveillance on potentially zoonotic viruses.

Durable sequence stability and bone marrow tropism in a macaque model of human pegivirus infection.

Human pegivirus (HPgV)-formerly known as GB virus C and hepatitis G virus-is a poorly characterized RNA virus that infects about one-sixth of the global human population and is transmitted frequently in the blood supply. We create an animal model of HPgV infection by infecting macaque monkeys with a new simian pegivirus (SPgV) discovered in wild baboons. Using this model, we provide a high-resolution, longitudinal picture of SPgV viremia where the dose, route, and timing of infection are known. We detail the highly variable acute phase of SPgV infection, showing that the viral load trajectory early in infection is dependent on the infecting dose, whereas the chronic-phase viremic set point is not. We also show that SPgV has an extremely low propensity for accumulating sequence variation, with no consensus-level variants detected during the acute phase of infection and an average of only 1.5 variants generated per 100 infection-days. Finally, we show that SPgV RNA is highly concentrated in only two tissues: spleen and bone marrow, with bone marrow likely producing most of the virus detected in plasma. Together, these results reconcile several paradoxical observations from cross-sectional analyses of HPgV in humans and provide an animal model for studying pegivirus biology.

Comparative single-cell genomics reveals potential ecological niches for the freshwater acI Actinobacteria lineage.

Members of the acI lineage of Actinobacteria are the most abundant microorganisms in most freshwater lakes; however, our understanding of the keys to their success and their role in carbon and nutrient cycling in freshwater systems has been hampered by the lack of pure cultures and genomes. We obtained draft genome assemblies from 11 single cells representing three acI tribes (acI-A1, acI-A7, acI-B1) from four temperate lakes in the United States and Europe. Comparative analysis of acI SAGs and other available freshwater bacterial genomes showed that acI has more gene content directed toward carbohydrate acquisition as compared to Polynucleobacter and LD12 Alphaproteobacteria, which seem to specialize more on carboxylic acids. The acI genomes contain actinorhodopsin as well as some genes involved in anaplerotic carbon fixation indicating the capacity to supplement their known heterotrophic lifestyle. Genome-level differences between the acI-A and acI-B clades suggest specialization at the clade level for carbon substrate acquisition. Overall, the acI genomes appear to be highly streamlined versions of Actinobacteria that include some genes allowing it to take advantage of sunlight and N-rich organic compounds such as polyamines, di- and oligopeptides, branched-chain amino acids and cyanophycin. This work significantly expands the known metabolic potential of the cosmopolitan freshwater acI lineage and its ecological and genetic traits.