Vaccine induction of CD4-mimicking HIV-1 broadly neutralizing antibody precursors in macaques.

The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.

Guiding HIV-1 vaccine development with preclinical nonhuman primate research.

PURPOSE OF THE REVIEW: Nonhuman primates (NHPs) are seen as the closest animal model to humans in terms of anatomy and immune system makeup. Here, we review how preclinical studies in this model system are teaching the field of HIV vaccinology the basic immunology that is needed to induce broadly neutralizing antibodies (bnAbs) with vaccination and elicit protective T cell responses. These lessons are being translated into clinical trials to advance towards protective active vaccination against HIV-1 infection. RECENT FINDINGS: Preclinical vaccination studies in NHPs have shown that highly engineered HIV-1 immunogens can initiate bnAb precursors providing proof of concept for Phase I clinical trials. Additionally, NHP models of HIV-1 infection are elucidating the pathways for bnAb development while serving as systems to evaluate vaccine protection. Innovative immunization strategies have increased affinity maturation of HIV-1 antibodies in long-lived germinal centers. Preclinical studies in macaques have defined the protective level of neutralizing antibodies and have shown that T cell responses can synergize with antibody-mediated immunity to provide protection in the presence of lower neutralizing antibody titers. SUMMARY: The NHP model provides vaccine regimens and desired antibody and T cell responses that serve as benchmarks for clinical trials, accelerating HIV vaccine design.

Fox Cluster determinants for iron biooxidation in the extremely thermoacidophilic Sulfolobaceae.

Within the extremely thermoacidophilic Sulfolobaceae, the capacity to oxidize iron varies considerably. While some species are prolific iron oxidizers (e.g. Metallosphaera sedula), other species do not oxidize iron at all (e.g. Sulfolobus acidocaldarius). Iron oxidation capacity maps to a genomic locus, referred to previously as the 'Fox Cluster', that encodes putative proteins that are mostly unique to the Sulfolobaceae. The role of putative proteins in the Fox Cluster has not been confirmed, but proteomic analysis here of iron-oxidizing membranes from M. sedula indicates that FoxA2 and FoxB (both cytochrome c oxidase-like subunits) and FoxC (CbsA/cytochrome b domain-containing) are essential. Furthermore, comparative genomics (locus organization and gene disruptions) and transcriptomics (polarity effects and differential expression) connect these genomic determinants with disparate iron biooxidation and respiration measurements among Sulfolobaceae species. While numerous homologous proteins can be identified for FoxA in genome databases (COX-like domains are prevalent across all domains of life), few homologues exist for FoxC or for most other Fox Cluster proteins. Phylogenetic reconstructions suggest this locus may have existed in early Sulfolobaceae, while the only other close homologues to the locus appear in the recently discovered candidate phylum Marsarchaota.

The biology of thermoacidophilic archaea from the order Sulfolobales.

Thermoacidophilic archaea belonging to the order Sulfolobales thrive in extreme biotopes, such as sulfuric hot springs and ore deposits. These microorganisms have been model systems for understanding life in extreme environments, as well as for probing the evolution of both molecular genetic processes and central metabolic pathways. Thermoacidophiles, such as the Sulfolobales, use typical microbial responses to persist in hot acid (e.g. motility, stress response, biofilm formation), albeit with some unusual twists. They also exhibit unique physiological features, including iron and sulfur chemolithoautotrophy, that differentiate them from much of the microbial world. Although first discovered >50 years ago, it was not until recently that genome sequence data and facile genetic tools have been developed for species in the Sulfolobales. These advances have not only opened up ways to further probe novel features of these microbes but also paved the way for their potential biotechnological applications. Discussed here are the nuances of the thermoacidophilic lifestyle of the Sulfolobales, including their evolutionary placement, cell biology, survival strategies, genetic tools, metabolic processes and physiological attributes together with how these characteristics make thermoacidophiles ideal platforms for specialized industrial processes.

Life in hot acid: a genome-based reassessment of the archaeal order Sulfolobales.

The order Sulfolobales was one of the first named Archaeal lineages, with globally distributed members from terrestrial thermal acid springs (pH < 4; T > 65°C). The Sulfolobales represent broad metabolic capabilities, ranging from lithotrophy, based on inorganic iron and sulfur biotransformations, to autotrophy, to chemoheterotrophy in less acidophilic species. Components of the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle, as well as sulfur oxidation, are nearly universally conserved, although dissimilatory sulfur reduction and disproportionation (Acidianus, Stygiolobus and Sulfurisphaera) and iron oxidation (Acidianus, Metallosphaera, Sulfurisphaera, Sulfuracidifex and Sulfodiicoccus) are limited to fewer lineages. Lithotrophic marker genes appear more often in highly acidophilic lineages. Despite the presence of facultative anaerobes and one confirmed obligate anaerobe, oxidase complexes (fox, sox, dox and a new putative cytochrome bd) are prevalent in many species (even facultative/obligate anaerobes), suggesting a key role for oxygen among the Sulfolobales. The presence of fox genes tracks with a putative antioxidant OsmC family peroxiredoxin, an indicator of oxidative stress derived from mixing reactive metals and oxygen. Extreme acidophily appears to track inversely with heterotrophy but directly with lithotrophy. Recent phylogenetic re-organization efforts are supported by the comparative genomics here, although several changes are proposed, including the expansion of the genus Saccharolobus.

Genome Sequences of Five Type Strain Members of the Archaeal Family Sulfolobaceae, Acidianus ambivalens, Acidianus infernus, Stygiolobus azoricus, Sulfuracidifex metallicus, and Sulfurisphaera ohwakuensis.

Presented are five genomes from the polyextremophilic (optimal temperature of >65°C and optimal pH of <3.5) archaeal family Sulfolobaceae, greatly expanding order-wide genomic diversity. Included are the only obligate anaerobic species, several facultative sulfur utilizers, two metal mobilizers, one facultative chemolithoautotroph with robust metabolic versatility, and some of the most thermophilic thermoacidophiles reported to date.

Determinants of sulphur chemolithoautotrophy in the extremely thermoacidophilic Sulfolobales.

Species in the archaeal order Sulfolobales thrive in hot acid and exhibit remarkable metabolic diversity. Some species are chemolithoautotrophic, obtaining energy through the oxidation of inorganic substrates, sulphur in particular, and acquiring carbon through the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) CO2 -fixation cycle. The current model for sulphur oxidation in the Sulfolobales is based on the biochemical analysis of specific proteins from Acidianus ambivalens, including sulphur oxygenase reductase (SOR) that disproportionates S° into H2 S and sulphite (SO3 2- ). Initial studies indicated SOR catalyses the essential first step in oxidation of elemental sulphur, but an ancillary role for SOR as a 'recycle' enzyme has also been proposed. Here, heterologous expression of both SOR and membrane-bound thiosulphate-quinone oxidoreductase (TQO) from Sulfolobus tokodaii 'restored' sulphur oxidation capacity in Sulfolobus acidocaldarius DSM639, but not autotrophy, although earlier reports indicate this strain was once capable of chemolithoautotrophy. Comparative transcriptomic analyses of Acidianus brierleyi, a chemolithoautotrophic sulphur oxidizer, and S. acidocaldarius DSM639 showed that while both share a strong transcriptional response to elemental sulphur, S. acidocaldarius DSM639 failed to upregulate key 3-HP/4-HB cycle genes used by A. brierleyi to drive chemolithoautotrophy. Thus, the inability for S. acidocaldarius DSM639 to grow chemolithoautotrophically may be rooted more in gene regulation than the biochemical capacity.

Extremely Thermoacidophilic Metallosphaera Species Mediate Mobilization and Oxidation of Vanadium and Molybdenum Oxides.

Certain species from the extremely thermoacidophilic genus Metallosphaera directly oxidize Fe(II) to Fe(III), which in turn catalyzes abiotic solubilization of copper from chalcopyrite to facilitate recovery of this valuable metal. In this process, the redox status of copper does not change as it is mobilized. Metallosphaera species can also catalyze the release of metals from ores with a change in the metal's redox state. For example, Metallosphaera sedula catalyzes the mobilization of uranium from the solid oxide U3O8, concomitant with the generation of soluble U(VI). Here, the mobilization of metals from solid oxides (V2O3, Cu2O, FeO, MnO, CoO, SnO, MoO2, Cr2O3, Ti2O3, and Rh2O3) was examined for M. sedula and M. prunae at 70°C and pH 2.0. Of these oxides, only V and Mo were solubilized, a process accelerated in the presence of FeCl3 However, it was not clear whether the solubilization and oxidation of these metals could be attributed entirely to an Fe-mediated indirect mechanism. Transcriptomic analysis for growth on molybdenum and vanadium oxides revealed transcriptional patterns not previously observed for growth on other energetic substrates (i.e., iron, chalcopyrite, organic compounds, reduced sulfur compounds, and molecular hydrogen). Of particular interest was the upregulation of Msed_1191, which encodes a Rieske cytochrome b6 fusion protein (Rcbf, referred to here as V/MoxA) that was not transcriptomically responsive during iron biooxidation. These results suggest that direct oxidation of V and Mo occurs, in addition to Fe-mediated oxidation, such that both direct and indirect mechanisms are involved in the mobilization of redox-active metals by Metallosphaera species.IMPORTANCE In order to effectively leverage extremely thermoacidophilic archaea for the microbially based solubilization of solid-phase metal substrates (e.g., sulfides and oxides), understanding the mechanisms by which these archaea solubilize metals is important. Physiological analysis of Metallosphaera species growth in the presence of molybdenum and vanadium oxides revealed an indirect mode of metal mobilization, catalyzed by iron cycling. However, since the mobilized metals exist in more than one oxidation state, they could potentially serve directly as energetic substrates. Transcriptomic response to molybdenum and vanadium oxides provided evidence for new biomolecules participating in direct metal biooxidation. The findings expand the knowledge on the physiological versatility of these extremely thermoacidophilic archaea.

Complete Genome Sequences of Extremely Thermoacidophilic Metal-Mobilizing Type Strain Members of the Archaeal Family Sulfolobaceae, Acidianus brierleyi DSM-1651, Acidianus sulfidivorans DSM-18786, and Metallosphaera hakonensis DSM-7519.

The family Sulfolobaceae contains extremely thermoacidophilic archaea that are found in terrestrial environments. Here, we report three closed genomes from two currently defined genera within the family, namely, Acidianus brierleyi DSM-1651T, Acidianus sulfidivorans DSM-18786T, and Metallosphaera hakonensis DSM-7519T.

Biotechnology of extremely thermophilic archaea.

Although the extremely thermophilic archaea (Topt ≥ 70°C) may be the most primitive extant forms of life, they have been studied to a limited extent relative to mesophilic microorganisms. Many of these organisms have unique biochemical and physiological characteristics with important biotechnological implications. These include methanogens that generate methane, fermentative anaerobes that produce hydrogen gas with high efficiency, and acidophiles that can mobilize base, precious and strategic metals from mineral ores. Extremely thermophilic archaea have also been a valuable source of thermoactive, thermostable biocatalysts, but their use as cellular systems has been limited because of the general lack of facile genetics tools. This situation has changed recently, however, thereby providing an important avenue for understanding their metabolic and physiological details and also opening up opportunities for metabolic engineering efforts. Along these lines, extremely thermophilic archaea have recently been engineered to produce a variety of alcohols and industrial chemicals, in some cases incorporating CO2 into the final product. There are barriers and challenges to these organisms reaching their full potential as industrial microorganisms but, if these can be overcome, a new dimension for biotechnology will be forthcoming that strategically exploits biology at high temperatures.

VapC toxins drive cellular dormancy under uranium stress for the extreme thermoacidophile Metallosphaera prunae.

When abruptly exposed to toxic levels of hexavalent uranium, the extremely thermoacidophilic archaeon Metallosphaera prunae, originally isolated from an abandoned uranium mine, ceased to grow, and concomitantly exhibited heightened levels of cytosolic ribonuclease activity that corresponded to substantial degradation of cellular RNA. The M. prunae transcriptome during 'uranium-shock' implicated VapC toxins as possible causative agents of the observed RNA degradation. Identifiable VapC toxins and PIN-domain proteins encoded in the M. prunae genome were produced and characterized, three of which (VapC4, VapC7, VapC8) substantially degraded M. prunae rRNA in vitro. RNA cleavage specificity for these VapCs mapped to motifs within M. prunae rRNA. Furthermore, based on frequency of cleavage sequences, putative target mRNAs for these VapCs were identified; these were closely associated with translation, transcription, and replication. It is interesting to note that Metallosphaera sedula, a member of the same genus and which has a nearly identical genome sequence but not isolated from a uranium-rich biotope, showed no evidence of dormancy when exposed to this metal. M. prunae utilizes VapC toxins for post-transcriptional regulation under uranium stress to enter a cellular dormant state, thereby providing an adaptive response to what would otherwise be a deleterious environmental perturbation.

Physiological, metabolic and biotechnological features of extremely thermophilic microorganisms.

The current upper thermal limit for life as we know it is approximately 120°C. Microorganisms that grow optimally at temperatures of 75°C and above are usually referred to as 'extreme thermophiles' and include both bacteria and archaea. For over a century, there has been great scientific curiosity in the basic tenets that support life in thermal biotopes on earth and potentially on other solar bodies. Extreme thermophiles can be aerobes, anaerobes, autotrophs, heterotrophs, or chemolithotrophs, and are found in diverse environments including shallow marine fissures, deep sea hydrothermal vents, terrestrial hot springs-basically, anywhere there is hot water. Initial efforts to study extreme thermophiles faced challenges with their isolation from difficult to access locales, problems with their cultivation in laboratories, and lack of molecular tools. Fortunately, because of their relatively small genomes, many extreme thermophiles were among the first organisms to be sequenced, thereby opening up the application of systems biology-based methods to probe their unique physiological, metabolic and biotechnological features. The bacterial genera Caldicellulosiruptor, Thermotoga and Thermus, and the archaea belonging to the orders Thermococcales and Sulfolobales, are among the most studied extreme thermophiles to date. The recent emergence of genetic tools for many of these organisms provides the opportunity to move beyond basic discovery and manipulation to biotechnologically relevant applications of metabolic engineering. WIREs Syst Biol Med 2017, 9:e1377. doi: 10.1002/wsbm.1377 For further resources related to this article, please visit the WIREs website.