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Vesicular stomatitis virus (VSV) is the most widely studied prototype for negative-sense RNA viruses. Structure determination of VSV particles is particularly challenging because they are polymorphic with different helical symmetries. Here, Jenni et al. apply computational classification approaches to sort different morphologies, and obtain CryoEM reconstructions at 3.5–4.1 A resolution. They show that the matrix protein (M) is present in the virion in two layers, of which the inner layer associates with N protein of vRNPs.

The conserved Ndc80 kinetochore complex, Ndc80c, is the principal link between mitotic spindle microtubules and centromere-associated proteins. We used AlphaFold 2 (AF2) to obtain predictions of the Ndc80 ‘loop’ structure and of the Ndc80 : Nuf2 globular head domains that interact with the Dam1 subunit of the heterodecameric DASH/Dam1 complex (Dam1c). The predictions guided design of crystallizable constructs, with structures close to the predicted ones. The Ndc80 ‘loop’ is a stiff, α-helical ‘switchback’ structure; AF2 predictions and positions of preferential cleavage sites indicate that flexibility within the long Ndc80c rod occurs instead at a hinge closer to the globular head. Conserved stretches of the Dam1 C terminus bind Ndc80c such that phosphorylation of Dam1 serine residues 257, 265 and 292 by the mitotic kinase Ipl1/Aurora B can release this contact during error correction of mis-attached kinetochores. We integrate the structural results presented here into our current molecular model of the kinetochore–microtubule interface. The model illustrates how multiple interactions between Ndc80c, DASH/Dam1c and the microtubule lattice stabilize kinetochore attachments.

ABSTRACT The first encounter with influenza virus biases later immune responses. This “immune imprinting,” formerly from infection within a few years of birth, is in the United States now largely from immunization with a quadrivalent, split vaccine (IIV4 [quadrivalent inactivated influenza vaccine]). In a pilot study of IIV4 imprinting, we used single-cell cultures, next-generation sequencing, and plasma antibody proteomics to characterize the primary antibody responses to influenza in two infants during their first 2 years of seasonal influenza vaccination. One infant, who received only a single vaccination in year 1, contracted an influenza B virus (IBV) infection between the 2 years, allowing us to compare imprinting by infection and vaccination. That infant had a shift in hemagglutinin (HA)-reactive B cell specificity from largely influenza A virus (IAV) specific in year 1 to IBV specific in year 2, both before and after the year 2 vaccination. HA-reactive B cells from the other infant maintained a more evenly distributed specificity. In year 2, class-switched HA-specific B cell IGHV somatic hypermutation (SHM) levels reached the average levels seen in adults. The HA-reactive plasma antibody repertoires of both infants comprised a relatively small number of antibody clonotypes, with one or two very abundant clonotypes. Thus, after the year 2 boost, both infants had overall B cell profiles that resembled those of adult controls. IMPORTANCE Influenza virus is a moving target for the immune system. Variants emerge that escape protection from antibodies elicited by a previously circulating variant (“antigenic drift”). The immune system usually responds to a drifted influenza virus by mutating existing antibodies rather than by producing entirely new ones. Thus, immune memory of the earliest influenza virus exposure has a major influence on later responses to infection or vaccination (“immune imprinting”). In the many studies of influenza immunity in adult subjects, imprinting has been from an early infection, since only in the past 2 decades have infants received influenza immunizations. The work reported in this paper is a pilot study of imprinting by the flu vaccine in two infants, who received the vaccine before experiencing an influenza virus infection. The results suggest that a quadrivalent (four-subtype) vaccine may provide an immune imprint less dominated by one subtype than does a monovalent infection.

An efficient HIV-1 vaccine will likely depend on eliciting broadly neutralizing antibodies (bnAb). Here the authors analyze the B cell repertoire in macaques and knock-in mice in response to sequential immunization with Env variants that induce a bnAb targeting the CD4-binding site of Env in a HIV-1 infected individual.

Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization.

Accurate segregation of genetic material in eukaryotes relies on the kinetochore, a multiprotein complex that connects centromeric DNA with microtubules. In yeast and humans, two proteins—Mif2/CENP-C and Chl4/CNEP-N—interact with specialized centromeric nucleosomes and establish distinct but cross-connecting axes of chromatin-microtubule linkage. Proteins recruited by Chl4/CENP-N include a subset that regulates chromosome transmission fidelity. We show that Chl4 and a conserved member of this subset, Iml3, both from Saccharomyces cerevisiae, form a stable protein complex that interacts with Mif2 and Sgo1. We have determined the structures of an Iml3 homodimer and an Iml3-Chl4 heterodimer, which suggest a mechanism for regulating the assembly of this functional axis of the kinetochore. We propose that at the core centromere, the Chl4-Iml3 complex participates in recruiting factors, such as Sgo1, that influence sister chromatid cohesion and encourage sister kinetochore biorientation.

The Saccharomyces cerevisiae monopolin complex directs proper chromosome segregation in meiosis I by mediating co-orientation of sister kinetochores on the meiosis I spindle. The monopolin subunits Csm1 and Lrs4 form a V-shaped complex that may directly crosslink sister kinetochores. We report here biochemical characterization of the monopolin complex subunits Mam1 and Hrr25 and of the complete four-protein monopolin complex. By purifying monopolin subcomplexes with different subunit combinations, we have determined the stoichiometry and overall architecture of the full monopolin complex. We have determined the crystal structure of Csm1 bound to a Mam1 fragment, showing how Mam1 wraps around the Csm1 dimer and alters the stoichiometry of kinetochore-protein binding by Csm1. We further show that the kinase activity of Hrr25 is altered by Mam1 binding, and we identify Hrr25 phosphorylation sites on Mam1 that may affect monopolin complex stability and/or kinetochore binding in meiosis.

Despite widespread yearly vaccination, influenza leads to significant morbidity and mortality across the globe. To make a more broadly protective influenza vaccine, it may be necessary to elicit antibodies that can activate effector functions in immune cells, such as antibody-dependent cellular cytotoxicity (ADCC). There is growing evidence supporting the necessity for ADCC in protection against influenza and herpes simplex virus (HSV), among other infectious diseases. An HSV-2 strain lacking the essential glycoprotein D (gD), was used to create ΔgD-2, which is a highly protective vaccine against lethal HSV-1 and HSV-2 infection in mice. It also elicits high levels of IgG2c antibodies that bind FcγRIV, a receptor that activates ADCC. To make an ADCC-eliciting influenza vaccine, we cloned the hemagglutinin ( HA ) gene from an H1N1 influenza A strain into the ΔgD-2 HSV vector. Vaccination with ΔgD-2::HA PR8 was protective against homologous influenza challenge and elicited an antibody response against HA that inhibits hemagglutination (HAI + ), is predominantly IgG2c, strongly activates FcγRIV, and protects against influenza challenge following passive immunization of naïve mice. Prior exposure of mice to HSV-1, HSV-2, or a replication-defective HSV-2 vaccine ( dl5-29 ) does not reduce protection against influenza by ΔgD-2::HA PR8 This vaccine also continues to elicit protection against both HSV-1 and HSV-2, including high levels of IgG2c antibodies against HSV-2. Mice lacking the interferon-α/β receptor and mice lacking the interferon-γ receptor were also protected against influenza challenge by ΔgD-2::HA PR8 Our results suggest that ΔgD-2 can be used as a vaccine vector against other pathogens, while also eliciting protective anti-HSV immunity., Competing Interests: Competing interest statement: The laboratories of W.R.J. receive financial support for sponsored research from X-Vax Technology, Inc., which holds licenses to several patents and patent applications related to ΔgD-2 vaccines, antibodies, and their use. W.R.J. serves as scientific advisor and consultant for the company. W.R.J. has equity interests in X-Vax Technology, Inc. W.R.J. is a coinventor on US patent no. 9,999,665 B2 “RECOMBINANT HERPES SIMPLEX VIRUS 2 (HSV-2) VACCINE VECTORS” and other patents related to ΔgD-2 vaccines, antibodies, and their use. D.M.K. is a coinventor on a patent on HSV-2 dl5-29 vaccine technology that is licensed by Harvard University to Sanofi Pasteur.