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news and v i ews of sequences increases with the addition of each sample so that as sequencing costs continue to fall, and the number of samples continues to increase, the accuracy of LSA should also increase10. Also, because LSA yields bins of sequencing reads, rather than clustering assembled sequences, different analysis approaches can be applied to its output, including approaches based both on de novo assembly9 and mapping11. De novo assembly can be used to generate new reference genomes of individual strains, whereas functional and taxonomic investigation techniques using mapping can be used to understand ecosystem functions of the community. The bins could also be clustered with other types of metadata, such as biochemical activity rates or virulence measures, potentially resolving the genomic sequence behind strain-specific phenotypes by correlating specific functional data with the abundance of sequence features across the samples. As it is a de novo preassembly approach, LSA avoids both the biases and computational challenges introduced by de novo assembly; here, the advantage is in both scaling analysis to larger data sets than de novo assembly can manage and avoiding the loss of sequence complexity involved in providing a single genomic sequence. Finally, LSA could be scaled to potentially hundreds of samples comprising terabytes of data, because it operates in fixed memory. De novo assembly methods

typically require specialized large-scale computers to run on terabase-size data sets such as those containing thousands of species; LSA should be able to run on the same data sets using much smaller computers. Cleary et al.1 explore many of the potential advantages of their pre-assembly algorithm. They demonstrate the ability of LSA to scale computationally by analyzing several extremely large data sets (300 Gb–4 Tb of data) using widely available computing hardware. Their largest analysis is of a data set that is tenfold larger than any reported previously. They also show that LSA can resolve population members that are present at abundances of only 1 in 10 million, although deep sequencing (50×) is required to observe such rare species. They show that LSA can resolve read bins to identify and separate reads that belong to closely related microbial strains if those strains vary in relative abundance across samples. Finally, they demonstrate that LSA can be tuned to coarser or finer resolution of samples, thereby enabling users to exert control over the final resolution of their data. Despite the substantial advances in sequence analysis that LSA enables, at least one significant challenge in basic metagenomic sequence analysis remains unsolved. Because different microbial strains may share large portions of chromosomes, along with complete plasmids and lysogenic phage, LSA on its own cannot group the core parts of genomes with all their

associated strain variants and will separate core modules into different partitions. New approaches will be needed to analyze combinations of strain-specific sequence and conserved features of pangenomes, which might be relevant for understanding community functions, although it may be possible to adapt existing strain variation approaches such as Platypus12. An improved ability to analyze large metagenomic sequencing data sets and better multivariate sampling approaches should advance the field of microbial ecology and improve our understanding of complex microbial communities. While it is too early to tell whether the LSA approach will be widely adopted, it has many advantages over current approaches and no obvious drawbacks. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Cleary, B. et al. Nat. Biotechnol. 33, 1053–1060 (2015). 2. Shakya, M. et al. Environ. Microbiol. 15, 1882–1899 (2013). 3. Sharon, I. et al. Genome Res. 23, 111–120 (2013). 4. Iverson, V. et al. Science 335, 587–590 (2012). 5. Bankevich, A. et al. J. Comput. Biol. 19, 455–477 (2012). 6. Abubucker, S. et al. PLoS Comput. Biol. 8, e1002358 (2012). 7. Alneberg, J. et al. Nat. Methods 11, 1144–1146 (2014). 8. Imelfort, M. et al. PeerJ 2, e603 (2014). 9. Pell, J. et al. Proc. Natl. Acad. Sci. USA 109, 13272–13277 (2012). 10. Pesant, S. et al. Sci Data 2, 150023 (2015). 11. Segata, N. et al. Nat. Methods 9, 811–814 (2012). 12. Rimmer, A. et al. Nat. Genet. 46, 912–918 (2014).

Stabilizing prospects for a universal flu vaccine Months before the flu season strikes each year, the World Health Organization (WHO; Geneva), the Centers for Disease Control (CDC; Atlanta) and other health organizations make a decision about which hemagglutinin (HA) and neuraminidase (NA) viral surface antigens to include in the vaccine design. The empirical nature of this process—essentially an educated guess based on epidemiological data from previous flu outbreaks and knowledge of the strains that are currently circulating—means that the effectiveness of conventional flu vaccines varies tremendously from year to year, depending on how well that vaccine matches the strain and lineage of influenza viruses that emerge. What’s more, the conventional flu vaccine is unlikely to offer protection against rapidly emerging pandemic virus strains that have no recent precedent, especially if these strains derive from serotypes other than the commonly circulating H1N1 and H3N2 subtypes included in the vaccine. For this reason, a flu vaccine that offers universal protection against all flu strains has remained the Holy Grail for the field. Two recent papers in

Science1 and Nature Medicine2 now present major steps toward the development of a flu vaccine that could offer protection against a much broader range of influenza strains across a variety of serotypes. Although flu vaccines comprise both HA and NA epitopes, antibodies against HA are thought to be particularly important in efficiently neutralizing the virus by blocking host cell attachment and entry. HA consists of two major domains: first, a globular head domain, which is highly divergent between different virus subtypes and strains, and, second, the stalk domain, which is much more conserved—and thus represents a more attractive target for a universal vaccine—but which rarely elicits a strong immune response in humans. The interest in stalk-reactive antibodies intensified when some were shown to bind and neutralize a broad range of HA types, suggesting that vaccines that trigger the production of such antibodies could offer much broader and potentially longerlasting protection against the ever-changing virus population circulating in the wild. That the human immune system is, in principle, able to make

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stalk-binding antibodies was verified in patients that survived infections with H5N1 (ref. 3) or the pandemic 2009 H1N1 (ref. 4) virus. It was quickly discovered, however, that the design of such vaccines poses daunting challenges. The conceptually easiest way to design a vaccine that elicits an immune response to the stalk region would be to remove the immunodominant globular head domain. But previous attempts to do this have resulted in conformational instability of the epitopes. The work by Impagliazzo et al.1 and Yassine et al.2 now shows that iterative protein engineering can yield HA versions that are both stable and able to elicit a protective immune response to heterosubtypic influenza challenges in animal models. Impagliazzo et al.1 use the HA of the of H1N1 A/Brisbane/59/2007 virus and develop their vaccine protein in a five-stage process. At the end of each stage, the most promising HA stalk constructs for further development were selected by measuring their affinity to two previously described broadly neutralizing antibodies. They replaced the head domain with a glycine linker, then introduced several hydrophilic

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amino acids to increase solubility and a cysteine bridge to increase stability. To preserve trimerization, they added the GCN4 leucine zipper sequence to the N-terminus of the protein. At the next stage, soluble versions were created by removing the transmembrane domain and various parts of the cytoplasmic domain. At stage III, a semi-rational library was screened to optimize the preservation of the relevant epitopes. At stage IV, the position of the GCN4 trimerization motif was optimized for epitope preservation and structural stability. Finally, at the last design stage, the stability of the trimeric construct was further increased by the introduction of intermolecular cysteine bridges. Only the final construct preserved the trimeric structure of HA in solution, whereas the best construct from stage IV primarily formed dimers and the other constructs, monomers. When tested in mice, the fully trimeric construct provided almost complete protection from a lethal challenge with a heterologous H1N1 virus, even after only one immunization. The other HA versions offered only partial protection after two or three inoculations. In a heterosubtypic setting, only constructs that were derived from the library optimization step (stage III and later) provided any protection, which increased with the multimerization level. Again, the fully trimeric form offered full protection from lethal challenge with an H5N1 virus. The

trimeric HA stalk vaccine was also tested in cynomolgus monkeys and, similarly to the seasonal flu vaccine, led to a substantial reduction in the disease symptoms of an H1N1 infection. In vitro assays showed that the antibodies produced in animals were able to both neutralize the virus directly (although this was tested only in H5N1-derived pseudoparticles) and induce antibody-dependent cellular cytotoxicity, which has been shown to be an important contributor to flu vaccine efficacy. Yassine et al.2 follow a similar strategy of structural optimization and selection of the best stalk constructs at each stage by broadly neutralizing antibody binding. They start with the ectodomain of the H1N1 A/New Caledonia/20/1999 virus they fused to the foldon trimerization domain. In subsequent generations, they engineer the following parts of the HA protein: the head domain is replaced by a short glycine-rich linker (1st generation); the membrane distal region is replaced by the HIV glycoprotein 41 (gp41) trimerization domain (2nd generation); the stem regions are further truncated (3rd generation); and finally linkers between HA and gp41 are optimized (4th generation). To avoid irrelevant immunogenicity, they subsequently replace the gp41 domains with linkers and add further core-stabilizing mutations (generations 5 and 6). Finally, to further increase the immunogenicity of the most promising constructs, they fused the ferritin subunit

of Helicobacter pylori to the engineered HA to create self-assembling nanoparticles. When mice and ferrets were immunized with these nanoparticles, a broad antibody response was elicited against various H1, H2, H3, H5, H7 and H9 influenza strains; however, in vitro neutralization activity was observed only for H1 strains. Nevertheless, the vaccine offered complete (mice) or partial (ferrets) protection from a lethal challenge with a heterosubtypic H5 virus. The molecular basis for this protection in the absence of direct virus neutralization activity is puzzling; the authors speculate that either antibody-dependent cell-mediated cytotoxicity or antibody-dependent, complement-mediated lysis might play a role, although these pathways have not been experimentally verified. Together these papers show that carefully optimized HA stalk constructs can be stable and immunogenic enough to elicit a strong antibody response to various HA serotypes, providing protection in three different animal species. How these concepts will translate to humans, of course, remains to be seen. 1. Impagliazzo, A. et al. Science 349, 1301–1306 (2015). 2. Yassine, H.M. et al. Nat. Med. 21, 1065–1070 (2015). 3. Kashyap, A. F. et al. Proc. Natl. Acad. Sci. USA 105, 5986–5991 (2008). 4. Pica, N. et al. Proc. Natl. Acad. Sci. USA 109, 2573–2578 (2012).

Research Highlights Papers from the literature selected by the Nature Biotechnology editors. (Follow us on Twitter, @NatureBiotech #nbtHighlight) Crystal structure of Staphylococcus aureus Cas9 Nishimasu, H. et al. Cell 162, 1113–1126 (2015) High-throughput pairing of T cell receptor a and b sequences Howie, B. et al. Sci. Transl. Med. 7, 131 (2015) Erosion of the chronic myeloid leukaemia stem cell pool by PPARg agonists Prost, S. et al. Nature 525, 380–383 (2015) Complete biosynthesis of opioids in yeast Galanie, S. et al. Science 349, 1095–1100 (2015) Genetic variance estimation with imputed variants finds negligible missing heritability for human height and body mass index Yang, J. et al. Nat. Genet. doi:10.1038/ng.3390 (31 August 2015)

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Stabilizing prospects for a universal flu vaccine.

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