Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.DOI: http://dx.doi.org/10.7554/eLife.23882.001

Highlights d Adenovirus vector-based vaccines protect pregnant mice against ZIKV challenge d Ad-based vaccines prevent ZIKV-induced intrauterine fetal growth restriction d Ad-based vaccines provide maternal-fetal protection d Passive transfer of vaccine-elicited antibodies protects infants from ZIKV challenge

Vaccine-elicited immunoglobulin G (IgG) has been shown to be important for protection against simian-human immunodeficiency virus (SHIV) infection in rhesus monkeys. However, it remains unclear whether vaccine-elicited IgA responses are beneficial or detrimental for protection. In this study, we evaluated the kinetics, magnitude, breadth, and linear epitope specificities of vaccine-elicited IgG and IgA responses in serum and mucosal secretions following intramuscular immunization with adenovirus 26 (Ad26) prime, Env protein boost vaccination regimens. The systemic and mucosal antibody responses exhibited kinetics similar to those of the serum antibody responses but lower titers than the serum antibody responses. Moreover, the IgG and IgA responses were correlated, both in terms of the magnitude of the responses and in terms of the antibody specificities against linear human immunodeficiency virus type 1 (HIV-1) Env, Gag, and Pol epitopes. These data suggest that IgG and IgA responses are highly coordinated in both peripheral blood and mucosal compartments following Ad26/Env vaccination in rhesus monkeys.IMPORTANCE Vaccine-elicited IgG responses are important for protection against simian-human immunodeficiency virus (SHIV) infection in nonhuman primates. However, much less is known about the role and function of IgA, despite it being the predominant antibody in mucosal sites. There is debate as to whether HIV-1-specific IgA responses are beneficial or detrimental, since serum anti-Env IgA titers were shown to be inversely correlated with protection in the RV144 clinical trial. We thus assessed vaccine-elicited IgG and IgA antibody responses in peripheral blood and mucosal secretions following vaccination with the Ad26/Env vaccine.

Adenovirus (Ad) vectors are being investigated as vaccine candidates, but baseline antivector immunity exists in human populations to both human Ad (HuAd) and chimpanzee Ad (ChAd) vectors. In this study, we investigated the immunogenicity and cross-reactivity of a panel of recently described rhesus adenoviral (RhAd) vectors. RhAd vectors elicited T cells with low exhaustion markers and robust anamnestic potential. Moreover, RhAd vector immunogenicity was unaffected by high levels of preexisting anti-HuAd immunity. Both HuAd/RhAd and RhAd/RhAd prime-boost vaccine regimens were highly immunogenic, despite a degree of cross-reactive neutralizing antibodies (NAbs) between phylogenetically related RhAd vectors. We observed extensive vector-specific cross-reactive CD4 T cell responses and more limited CD8 T cell responses between RhAd and HuAd vectors, but the impact of vector-specific cellular responses was far less than that of vector-specific NAbs. These data suggest the potential utility of RhAd vectors and define novel heterologous prime-boost strategies for vaccine development.IMPORTANCE To date, most adenoviral vectors developed for vaccination have been HuAds from species B, C, D, and E, and human populations display moderate to high levels of preexisting immunity. There is a clinical need for new adenoviral vectors that are not hindered by preexisting immunity. Moreover, the development of RhAd vector vaccines expands our ability to vaccinate against multiple pathogens in a population that may have received other HuAd or ChAd vectors. We evaluated the immunogenicity and cross-reactivity of RhAd vectors, which belong to the poorly described adenovirus species G. These vectors induced robust cellular and humoral immune responses and were not hampered by preexisting anti-HuAd vector immunity. Such properties make RhAd vectors attractive as potential vaccine vectors.

Prime-boost immunization regimens have proven efficacious at generating robust immune responses. However, whether the level of replication of the boosting antigen impacts the magnitude and protective efficacy of vaccine-elicited immune responses remains unclear. To evaluate this, we primed mice with replication-defective adenovirus vectors expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP), followed by boosting with either LCMV Armstrong, which is rapidly controlled, or LCMV CL-13, which leads to a more prolonged exposure to the boosting antigen. Although priming of naive mice with LCMV CL-13 normally results in T cell exhaustion and establishment of chronic infection, boosting with CL-13 resulted in potent recall CD8 T cell responses that were greater than those following boosting with LCMV Armstrong. Furthermore, following the CL-13 boost, a greater number of anamnestic CD8 T cells localized to the lymph nodes, exhibited granzyme B expression, and conferred improved protection against Listeria and vaccinia virus challenges compared with the Armstrong boost. Overall, our findings suggest that the replicative capacity of the boosting antigen influences the protective efficacy afforded by prime-boost vaccine regimens. These findings are relevant for optimizing vaccine candidates and suggest a benefit of robustly replicating vaccine vectors. IMPORTANCEThe development of optimal prime-boost vaccine regimens is a high priority for the vaccine development field. In this study, we compared two boosting antigens with different replicative capacities. Boosting with a more highly replicative vector resulted in augmented immune responses and improved protective efficacy.A major challenge in the development of T cell-based vaccines is the generation of CD8 T cell responses of sufficient quantity and quality. Following immunization, cytotoxic CD8 T cells undergo extensive division and differentiation into long-lived memory cells. The number and function of antigen-specific T cells are influenced by several factors, including the route of antigen delivery (1), distinct triggering of innate responses (2-4), the degree of costimulation (5-10), the prime-boost time interval (11,12), the level of preexisting immunity to the vaccine vector (13-15), and the recall history of responding T cells (16)(17)(18). Although the magnitude and duration of the priming stimulus have been shown to impact the function, numbers, and differentiation of CD8 T cells (1,(19)(20)(21)(22)(23)(24)(25)(26)(27), it is currently not known how the nature of the boosting stimulus influences anamnestic responses and immune protection by memory CD8 T cells.Alternative serotype adenovirus (Ad) vectors, such as adenovirus type 26 (Ad26) and Ad35, have been shown to induce functionally improved memory T cell responses than that induced by Ad5 (11,28). We have also demonstrated the protective efficacy of vaccine regimens based on alternative serotype Ad vectors against high-dose intravenous (i.v.) and repetitive low-dose intrarecta...

bHuman immunodeficiency virus type 1 (HIV-1) vaccines that elicit protective antibody responses at mucosal sites would be highly desirable. Here, we report that intramuscular immunization of candidate HIV-1 vaccine vectors and purified Env proteins elicited potent and durable humoral immune responses in colorectal mucosa in rhesus monkeys. The kinetics, isotypes, functionality, and epitope specificity of these mucosal antibody responses were similar to those of peripheral responses in serum. These data suggest a close immunological relationship between mucosal and systemic antibody responses following vaccination in primates. Human mucosal surfaces represent the major portal of entry for human immunodeficiency virus type 1 (HIV-1) (1). Thus, a prophylactic vaccine will likely need to elicit protective antibody responses at mucosal sites of virus exposure. However, mucosal humoral immune responses following vaccination are poorly characterized. The RV144 clinical trial suggested that vaccine-elicited HIV-1 envelope (Env)-specific humoral immune responses may have contributed to the partial protection observed for vaccinees (2), but mucosal immune responses were not assessed in that trial. In contrast, previous studies have shown that immunization with peptide, DNA, protein, or attenuated bacterial or viral vector-based vaccines through parental or mucosal routes may elicit antigen-specific humoral immune responses at mucosal sites in mice, nonhuman primates, and humans (3-7). However, the characteristics, functionality, and epitope specificity of vaccine-elicited mucosal antibody responses have not been fully explored. Moreover, whether mucosal antibody responses reflect distinct populations compared with those for peripheral antibody responses remains to be determined. We therefore assessed the magnitude, durability, isotype, neutralizing activity, and epitope specificity of mucosal and peripheral antibody responses in rhesus monkeys elicited by adenovirus (Ad) vector-based and proteinbased HIV-1 vaccine candidates.We first collected blood and colorectal mucosal secretions using Weck-Cel sponges from 8 healthy adult rhesus monkeys. Using sera and mucosal secretions eluted from Weck-Cel sponges (8), we assessed the amount of total IgG and IgA (monkey IgG/IgA enzyme-linked immunosorbent assay [ELISA] kit; Alpha Diagnostic). The average volume of eluates from 8 unused sponges was used as the elution buffer volume, and a dilution factor was calculated according to the volume of eluate for each sample: dilution factor ϭ experiment sponge eluate volume/(experiment sponge eluate volume Ϫ unused sponge eluate volume). The dilution factor was used to calculate total IgG and IgA as well as titers

Natural killer (NK) cells respond rapidly as a first line of defense against infectious pathogens. In addition, NK cells may provide a “rheostat” function and have been shown to reduce the magnitude of antigen-specific T cell responses following infection to avoid immunopathology. However, it remains unknown whether NK cells similarly modulate vaccine-elicited T cell responses following virus challenge. We used the lymphocytic choriomeningitis virus (LCMV) clone 13 infection model to address whether NK cells regulate T cell responses in adenovirus vector-vaccinated mice following challenge. As expected, NK cell depletion in unvaccinated mice resulted in increased virus-specific CD4+ and CD8+ T cell responses and immunopathology following LCMV challenge. In contrast, NK cell depletion had minimal to no impact on antigen-specific T cell responses in mice that were vaccinated with an adenovirus serotype 5 (Ad5)-GP vector prior to LCMV challenge. Moreover, NK cell depletion in vaccinated mice prior to challenge did not result in immunopathology and did not compromise protective efficacy. These data suggest that adenovirus vaccine-elicited T cells may be less sensitive to NK cell rheostat regulation than T cells primed by LCMV infection.IMPORTANCE Recent data have shown that NK cell depletion leads to enhanced virus-elicited T cell responses that can result in severe immunopathology following LCMV infection in mice. In this study, we observed that NK cells exerted minimal to no impact on vaccine-elicited T cells following LCMV challenge, suggesting that adenovirus vaccine-elicited T cells may be less subject to NK cell regulation. These data contribute to our understanding of NK cell regulatory functions and T cell-based vaccines.