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mRNA lipid nanoparticle (LNP) vaccines would be useful during an influenza virus pandemic since they can be produced rapidly and do not require the generation of egg-adapted vaccine seed stocks. Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. Here, we generate an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. The H5 mRNA-LNP vaccine elicits strong T cell and antibody responses in female mice, including neutralizing antibodies and broadly-reactive anti-HA stalk antibodies. The H5 mRNA-LNP vaccine elicits antibodies at similar levels compared to whole inactivated vaccines in female mice with and without prior H1N1 exposures. Finally, we find that the H5 mRNA-LNP vaccine is immunogenic in male ferrets and prevents morbidity and mortality of animals following 2.3.4.4b H5N1 challenge. Together, our data demonstrate that a monovalent mRNA-LNP vaccine expressing 2.3.4.4b H5 is immunogenic and protective in pre-clinical animal models. Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating widely in birds and have recently caused large outbreaks in mammals. Here, Furey et al. develop a clade 2.3.4.4b HA-expressing mRNA-LNP vaccine and show that it elicits strong protective immune responses in mice and ferrets.

H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple nonhuman mammalian species. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic potential in humans, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. However, prior experimental work has only measured how these phenotypes are affected by a handful of the >10,000 different possible amino-acid mutations to HA. Here, we use pseudovirus deep mutational scanning to measure how all mutations to a 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind α2-6-linked sialic acids and show that some viruses already carry mutations that stabilize HA. We also measure how all HA mutations affect neutralization by sera from mice and ferrets vaccinated against or infected with 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired mutations that may create mismatches with candidate vaccine virus, and we show that a mutation present in some recent H5 HAs causes a large antigenic change. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive measurements of the phenotypic effects of mutations that can inform real-time interpretation of viral variation observed during surveillance of H5 influenza.

The chikungunya virus (CHIKV) is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates with the introduction of its major vector, Aedes albopictus. CHIKV causes a disease frequently misdiagnosed as dengue fever, with potentially life-threatening symptoms that can result in a longer-term debilitating arthritis. The increasing risk of spread from endemic regions via human travel and commerce and the current absence of a vaccine put a significant proportion of the world population at risk for this disease. In this study we designed and tested hammerhead ribozymes (hRzs) targeting CHIKV structural protein genes of the RNA genome as potential antivirals both at the cellular and in vivo level. We employed the CHIKV strain 181/25, which exhibits similar infectivity rates in both Vero cell cultures and mosquitoes. Virus suppression assay performed on transformed Vero cell clones of all seven hRzs demonstrated that all are effective at inhibiting CHIKV in Vero cells, with hRz #9 and #14 being the most effective. piggyBac transformation vectors were constructed using the Ae. aegypti t-RNAval Pol III promoted hRz #9 and #14 effector genes to establish a total of nine unique transgenic Higgs White Eye (HWE) Ae. aegypti lines. Following confirmation of transgene expression by real-time polymerase chain reaction (RT-PCR), comparative TCID50-IFA analysis, in situ Immuno-fluorescent Assays (IFA) and analysis of salivary CHIKV titers demonstrated effective suppression of virus replication at 7 dpi in heterozygous females of each of these transgenic lines compared with control HWE mosquitoes. This report provides a proof that appropriately engineered hRzs are powerful antiviral effector genes suitable for population replacement strategies

Phages infecting bacteria of the genus Staphylococcus play an important role in their host’s ecology and evolution. On one hand, horizontal gene transfer from phage can encourage the rapid adaptation of pathogenic Staphylococcus enabling them to escape host immunity or access novel environments. On the other hand, lytic phages are promising agents for the treatment of bacterial infections, especially those resistant to antibiotics. As part of an ongoing effort to gain novel insights into bacteriophage diversity, we characterized the complete genome of the Staphylococcus bacteriophage Metroid, a cluster C phage with a genome size of 151kb, encompassing 254 predicted protein-coding genes as well as 4 tRNAs. A comparative genomic analysis highlights strong similarities – including a conservation of the lysis cassette – with other Staphylococcus cluster C bacteriophages, several of which were previously characterized for therapeutic applications.

Despite its size and rigidity, the cell nucleus can be moved or reorganized by cytoskeletal filaments under various conditions (for example, during viral infection) 1 – 11 . Moreover, whereas chromatin organizes into non-random domains 12 , extensive heterogeneity at the single-cell level 13 means that precisely how and why nuclei reorganize remains an area of intense investigation. Here we describe convolutional neural network-based automated cell classification and analysis pipelines, which revealed the extent to which human cytomegalovirus generates nuclear polarity through a virus-assembled microtubule-organizing centre. Acetylation of tubulin enables microtubules emanating from this centre to rotate the nucleus by engaging cytoplasmically exposed dynein-binding domains in the outer nuclear membrane protein nesprin-2G, which polarizes the inner nuclear membrane protein SUN1. This in turn creates intranuclear polarity in emerin, and thereby controls nuclear actin filaments that spatially segregate viral DNA from inactive histones and host DNA, maximizing virus replication. Our findings demonstrate the extent to which viruses can control the nucleus from the cytoplasm. Human cytomegalovirus rotates the nuclei of infected cells to set up intranuclear polarization and thereby separate viral DNA from inactive histones and associated host DNA.

Avian H5N1 influenza viruses are circulating widely in cattle and other mammals and pose a risk for a human pandemic. Previous studies suggest that older humans are more resistant to H5N1 infections due to childhood imprinting with other group 1 viruses (H1N1 and H2N2); however, the immunological basis for this is incompletely understood. Here we measured H5N1 antibody responses in sera from 157 individuals born between 1927 and 2016. We show that antibody titers to historical and recent H5N1 strains are highest in older individuals and correlate more strongly with birth year than with age, consistent with immune imprinting. Young children, who were likely not yet exposed to seasonal influenza viruses, had low levels of H5-specific antibodies. We also measured H5N1 antibody responses in sera from 100 individuals before and after receiving an A/Vietnam/1203/2004 H5N1 vaccine. We found that both younger and older humans produced H5-reactive antibodies to the A/Vietnam/1203/2004 vaccine strain and to a contemporary clade 2.3.4.4b strain, with higher seroconversion rates in young children who had lower levels of antibodies before vaccination. These studies suggest that younger individuals might benefit more from vaccination than older individuals in the event of an H5N1 pandemic. H5N1 strain-specific antibodies are higher in older individuals and correlate more with birth year than with age, suggesting that younger individuals are potentially more likely to benefit from H5N1 vaccination.

The microtubule (MT) network and associated regulatory proteins play a critical role during viral infection from facilitating viral particle transport towards the nucleus upon entry to later mediating virion assembly and egress. Many of the precise mechanisms by which viruses commandeer the host MT network to propagate infection remain poorly defined. The MT network is largely controlled by a group of proteins known as plus-end tracking proteins or +TIPs which directly or indirectly bind growing MT plus-ends and regulate their dynamic instability. Among the +TIPs, the end-binding (EB) proteins have long been considered the master regulators of the MT network due to their ability to directly bind MT plus-ends and recruit a diverse array of other +TIP partners there. As mediators of MT dynamics and stability, +TIPs can be exploited by viruses to manipulate MT behavior. Indeed, previous work from our lab demonstrated that herpes simplex virus type 1 (HSV-1) hijacks a +TIP complex including EB1 at the cell periphery to initiate viral particle transport on dynamic tyrosinated MTs during early infection in primary normal human dermal fibroblasts (NHDFs). In examining whether HSV-1 utilizes similar mechanisms during infections in other cell types, I found that HSV-1 adopts a different approach to infect the neuronal SK-N-SH cell line, with HSV-1 particles trafficking upon stable de-tyrosinated MTs in an EB-independent manner to the nucleus. To explore the potential contribution of an EB-independent +TIP to HSV-1 infection in SK-N-SHs, I began studying transforming acidic coiled-coil protein 3 (TACC3), an autonomous tip-tracker primarily characterized as a mitotic protein that promotes MT spindle elongation by recruiting the MT polymerase chTOG to spindle plus-ends. Through my studies, I found that TACC3 plays a role not only in facilitating HSV-1 infection in both NHDFs and SK-N-SHs, but more generally in controlling interphase MT dynamics. In this work, I show that altering TACC3 levels disrupts the nuclear-cytoplasmic localization of chTOG and thus the balance between dynamic and stable MTs in the cell. Loss of TACC3 results in nuclear sequestration of chTOG, reduced MT growth in the cytoplasm, and accumulation of post-translationally modified stable MTs. In NHDFs, the loss of EB1 and dynamic MTs at the periphery of TACC3-depleted cells inhibits HSV-1 particle transport to the nucleus and subsequently blocks infection. In SK-N-SHs the upregulation of de-tyrosinated MTs in TACC3-depleted cells results in impaired HSV-1 particle transport to the nucleus which I found is due to the biasing of MT-dependent transport towards outward-directing kinesin motors. Together, these findings highlight the previously unappreciated role for TACC3 in regulating interphase MTs and in turn facilitating transport of cellular and viral cargo. To expand upon these findings, I next examined the role of TACC3 in a different herpesvirus infection, human cytomegalovirus (HCMV). Compared to HSV-1, HCMV has a protracted infectious cycle and is characterized by the formation of a viral assembly compartment (AC) which consists of rearranged host secretory machinery and requires precise coordination by the MT network. I found that HCMV specifically upregulates TACC3 late in infection to maintain high levels of chTOG and dynamic MTs in the cytoplasm, which is necessary for recruitment of secretory machinery to the AC and subsequent virion egress. The work presented here provides evidence towards the centrality of TACC3 in MT network regulation. This work also presents an example of a virus targeting TACC3 to promote infection, laying a foundation for future investigations of TACC3 in the context of other viral infections.

H5 influenza is a potential pandemic threat. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic risk, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. Here we use pseudovirus deep mutational scanning to measure how all mutations to a clade 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind a2-6-linked sialic acids, and show that some viruses already carry mutations that stabilize HA. We also identify recent viral strains with reduced neutralization to sera elicited by candidate vaccine virus. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive characterization of mutations to inform surveillance of H5 influenza.

Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. We generated an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. We show that the vaccine is immunogenic in mice and ferrets and prevents morbidity and mortality of ferrets following 2.3.4.4b H5N1 challenge.Highly pathogenic avian influenza viruses from H5 clade 2.3.4.4b are circulating at unprecedently high levels in wild and domestic birds and have the potential to adapt to humans. We generated an mRNA lipid nanoparticle (LNP) vaccine encoding the hemagglutinin (HA) glycoprotein from a clade 2.3.4.4b H5 isolate. We show that the vaccine is immunogenic in mice and ferrets and prevents morbidity and mortality of ferrets following 2.3.4.4b H5N1 challenge.