The emergence of pandemic influenza viruses poses a major public health threat. Therefore, there is a need for a vaccine that can induce broadly cross-reactive antibodies that protect against seasonal as well as pandemic influenza strains. Human broadly neutralizing antibodies directed against highly conserved epitopes in the stem region of influenza virus HA have been recently characterized. However, it remains unknown what the baseline levels are of antibodies and memory B cells that are directed against these conserved epitopes. More importantly, it is also not known to what extent anti-HA stem B-cell responses get boosted in humans after seasonal influenza vaccination. In this study, we have addressed these two outstanding questions. Our data show that: (i) antibodies and memory B cells directed against the conserved HA stem region are prevalent in humans, but their levels are much lower than B-cell responses directed to variable epitopes in the HA head; (ii) current seasonal influenza vaccines are efficient in inducing B-cell responses to the variable HA head region but they fail to boost responses to the conserved HA stem region; and (iii) in striking contrast, immunization of humans with the avian influenza virus H5N1 induced broadly cross-reactive HA stem-specific antibodies. Taken together, our findings provide a potential vaccination strategy where heterologous influenza immunization could be used for increasing the levels of broadly neutralizing antibodies and for priming the human population to respond quickly to emerging pandemic influenza threats. Significance Vaccination is the most effective means of attaining protection against influenza viruses. However, the constantly evolving nature of influenza viruses enables them to escape preexisting immune surveillance, and thus thwarts public health efforts to control influenza annual epidemics and occasional pandemics. One solution is to elicit antibodies directed against highly conserved epitopes, such as those within the stem region of influenza HA, the principal target of virus-neutralizing antibody responses. This study shows that annual influenza vaccines induce antibody responses that are largely directed against the highly variable HA head region. In contrast, heterologous immunization with HA derived from influenza strains that are currently not circulating in humans (e.g. H5N1) can substantially increase HA stem-specific responses.

Tetherin/BST2 was identified in 2008 as the cellular factor responsible for restricting HIV-1 replication at a very late stage in the lifecycle. Tetherin acts to retain virion particles on the plasma membrane after budding has been completed. Infected cells that express large amounts of tetherin display large strings of HIV virions that remain attached to the plasma membrane. Vpu is an HIV-1 accessory protein that specifically counteracts the restriction to virus release contributed by tetherin. Tetherin is an unusual Type II transmembrane protein that contains a GPI anchor at its C-terminus and is found in lipid rafts. The leading model for the mechanism of action of tetherin is that it functions as a direct physical tether bridging virions and the plasma membrane. However, evidence that tetherin functions as a physical tether has thus far been indirect. Here we demonstrate by biochemical and immunoelectron microscopic methods that endogenous tetherin is present on the viral particle and forms a bridge between virion particles and the plasma membrane. Endogenous tetherin was found on HIV particles that were released by partial proteolytic digestion. Immunoelectron microscopy performed on HIV-infected T cells demonstrated that tetherin forms an apparent physical link between virions and connects patches of virions to the plasma membrane. Linear filamentous strands that were highly enriched in tetherin bridged the space between some virions. We conclude that tetherin is the physical tether linking HIV-1 virions and the plasma membrane. The presence of filaments with which multiple molecules of tetherin interact in connecting virion particles is strongly suggested by the morphologic evidence. Tetherin or BST2 is a cellular protein that was recently found to limit the ability of HIV to escape from cells. HIV counteracts this cellular restriction to its lifecycle by expressing the small viral accessory protein Vpu. Upon viral infection, cells expressing high levels of tetherin accumulate large clusters or strings of virions that remain attached to the plasma membrane by an unknown mechanism. The simplest explanation for this clustering is that tetherin itself physically attaches particles to the plasma membrane and to each other. In this article, we demonstrate that this is indeed the case. We found that particles released from cells by gentle protease treatment contain either cleaved or full-length tetherin. Using electron microscopy and immunogold staining, we show that tetherin is present as a physical link between viruses and the plasma membrane and sometimes between virus particles in large clusters or strings. Together this provides evidence that tetherin serves a direct, physical role in retaining particles on the surface of cells.

In 2013, human infections with a novel influenza subtype, A/H7N9, were identified in China.¹ Infected patients had live poultry exposures. The virus produced no disease in poultry; however, it caused severe pneumonia in humans, with hospitalization and mortality rates of 67% and 33%, respectively.^1,2 Limited human-to-human spread of A/H7N9 in households was documented³ without sustained transmission. A second wave of infections occurred in winter 2013-2014; through June 27, 2014, a total of 450 laboratory-confirmed cases with 165 deaths (36.6%) were reported to the World Health Organization.⁴

HIV-1 particles assemble and bud from the plasma membrane of infected T lymphocytes. Infected macrophages, in contrast, accumulate particles within an apparent intracellular compartment known as the virus-containing compartment or VCC. Many aspects of the formation and function of the VCC remain unclear. Here we demonstrate that VCC formation does not actually require infection of the macrophage, but can be reproduced through the exogenous addition of non-infectious virus-like particles or infectious virions to macrophage cultures. Particles were captured by Siglec-1, a prominent cell surface lectin that attaches to gangliosides on the lipid envelope of the virus. VCCs formed within infected macrophages were readily targeted by the addition of ganglioside-containing virus-like particles to the extracellular media. Depletion of Siglec-1 from the macrophage or depletion of gangliosides from viral particles prevented particle uptake into the VCC and resulted in substantial reductions of VCC volume. Furthermore, Siglec-1-mediated virion capture and subsequent VCC formation was required for efficient trans-infection of autologous T cells. Our results help to define the nature of this intracellular compartment, arguing that it is a compartment formed by particle uptake from the periphery, and that this compartment can readily transmit virus to target T lymphocytes. Inhibiting or eliminating the VCC may be an important component of strategies to reduce HIV transmission and to eradicate HIV reservoirs. T lymphocytes and macrophages are the two major cell types involved in HIV replication and transmission events. When a T cell is infected, virus particles assemble and bud from the plasma membrane of the cell. In contrast, infected macrophages develop an intracellular collection of viruses termed the virus-containing compartment or VCC. Many aspects of the formation and function of the VCC remain unclear. Here we show that VCC formation does not actually require infection of the macrophage, but can be reproduced through the addition of virus-like particles or infectious virions to macrophages. HIV-1 particles were captured by the cell surface carbohydrate-binding protein Siglec-1, followed by co-migration of Siglec-1 and captured viral particles to the VCC. Depletion of Siglec-1 from the macrophage prevented VCC formation, and inhibited the ability of infected macrophages to transmit HIV to T cells. Our results help to define the origin of this intracellular compartment, arguing that it is a compartment formed by particle uptake from the periphery. Inhibiting or eliminating the VCC may be an important component of strategies to reduce HIV transmission and to eradicate HIV reservoirs.