Accumulation of activated natural killer (NK) cells in tissues during Ebola virus infection contributes to Ebola virus disease (EVD) pathogenesis. Yet, immunization with Ebola virus-like particles (VLPs) comprising glycoprotein and matrix protein VP40 provides rapid, NK cell-mediated protection against Ebola challenge. We used Ebola VLPs as the viral surrogates to elucidate the molecular mechanism by which Ebola virus triggers heightened NK cell activity. Incubation of human peripheral blood mononuclear cells with Ebola VLPs or VP40 protein led to increased expression of IFN-γ, TNF-α, granzyme B, and perforin by CD3-CD56+ NK cells, along with increases in degranulation and cytotoxic activity of these cells. Optimal activation required accessory cells like CD14+ myeloid and CD14- cells and triggered increased secretion of numerous inflammatory cytokines. VP40-induced IFN-γ and TNF-α secretion by NK cells was dependent on IL-12 and IL-18 and suppressed by IL-10. In contrast, their increased degranulation was dependent on IL-12 with little influence of IL-18 or IL-10. These results demonstrate that Ebola VP40 stimulates NK cell functions in an IL-12- and IL-18-dependent manner that involves CD14+ and CD14- accessory cells. These potentially novel findings may help in designing improved intervention strategies required to control viral transmission during Ebola outbreaks.

Electron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.

Ebolaviruses are highly virulent pathogens that cause Ebola viral disease (EVD). Data from non-human primate (NHP) models and from human survivors of EVD suggest that anti-Ebola antibodies play an integral role in protection. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a potential mechanism through which anti-Ebola antibodies may mediate protection. We developed a robust Ebola-specific ADCC assay for use in ongoing trials of Ebola vaccines. Stable cell lines for inducible Zaire ebolavirus glycoprotein (EBOV GP) expression were developed to provide a uniform source of target cells in the assay, and were combined with an existing human natural killer (NK) cell line as the effector cell. When applied to commercially available anti-EBOV GP monoclonal antibodies, the assay clearly differentiated antibody with high ADCC activity from those with low or no ADCC activity. Anti-EBOV ADCC activity was also detected in plasma samples from rhesus macaques immunized with a candidate Ebola vaccine. The Ebola ADCC assay reported here will be a useful tool in studying the functionality of anti-EBOV GP antibodies elicited by Ebola vaccines in ongoing and future clinical trials.

The intracellular transport of Mason-Pfizer monkey virus (M-PMV) assembled capsids from the pericentriolar region to the plasma membrane (PM) requires trafficking of envelope glycoprotein (Env) to the assembly site via the recycling endosome. However, it is unclear if Env-containing vesicles play a direct role in trafficking capsids to the PM. Using live cell microscopy, we demonstrate, for the first time, anterograde co-transport of Gag and Env. Nocodazole disruption of microtubules had differential effects on Gag and Env trafficking, with pulse-chase assays showing a delayed release of Env-deficient virions. Particle tracking demonstrated an initial loss of linear movement of GFP-tagged capsids and mCherry-tagged Env, followed by renewed movement of Gag but not Env at 4. h post-treatment. Thus, while delayed capsid trafficking can occur in the absence of microtubules, efficient anterograde transport of capsids appears to be mediated by microtubule-associated Env-containing vesicles.

Background: Despite anti-retroviral therapy, HIV-1 infection increases the risk of pneumonia and causes oxidative stress and defective alveolar macrophage (AM) immune function. We have previously determined that HIV-1 proteins inhibit antioxidant defenses and impair AM phagocytosis by suppressing nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Given its known effects on Nrf2, we hypothesize miR-144 mediates the HIV-1 induced suppression of Nrf2. Methods: Primary AMs isolated from HIV-1 transgenic (HIV-1 Tg) rats and wild type littermates (WT) as well as human monocyte-derived macrophages (MDMs) infected ex vivo with HIV-1 were used. We modulated miR-144 expression using a miR-144 mimic or an inhibitor to assay its effects on Nrf2/ARE activity and AM functions in vitro and in vivo. Results: MiR-144 expression was increased in AMs from HIV-1 Tg rats and in HIV-1-infected human MDMs compared to cells from WT rats and non-infected human MDMs, respectively. Increasing miR-144 with a miR-144 mimic inhibited the expression of Nrf2 and its downstream effectors in WT rat macrophages and consequently impaired their bacterial phagocytic capacity and H2O2 scavenging ability. These effects on Nrf2 expression and AM function were reversed by antagonizing miR-144 ex vivo or in the airways of HIV-1 Tg rats in vivo, but this protection was abrogated by silencing Nrf2 expression. Conclusions: Our results suggest that inhibiting miR-144 or interfering with its deleterious effects on Nrf2 attenuates HIV-1-mediated AM immune dysfunction and improves lung health in individuals with HIV.

Our previous work has shown that antigens adjuvanted with ligands specific for Toll-like receptor 4 (TLR4) and TLR7/8 encapsulated in poly(lactic-co-glycolic) acid (PLGA)-based nanoparticles (NPs) induce robust and durable immune responses in mice and macaques. We investigated the efficacy of these NP adjuvants in inducing protective immunity against simian immunodeficiency virus (SIV). Rhesus macaques (RMs) were immunized with NPs containing TLR4 and TLR7/8 agonists mixed with soluble recombinant SIVmac239-derived envelope (Env) gp140 and Gag p55 (protein) or with virus-like particles (VLPs) containing SIVmac239 Env and Gag. NPadjuvanted vaccines induced robust innate responses, antigen-specific antibody responses of a greater magnitude and persistence, and enhanced plasmablast responses compared to those achieved with alum-adjuvanted vaccines. NP-adjuvanted vaccines induced antigen-specific, long-lived plasma cells (LLPCs), which persisted in the bone marrow for several months after vaccination. NP-adjuvanted vaccines induced immune responses that were associated with enhanced protection against repeated low-dose, intravaginal challenges with heterologous SIVsmE660 in animals that carried TRIM5α restrictive alleles. The protection induced by immunization with protein-NP correlated with the prechallenge titers of Env-specific IgG antibodies in serum and vaginal secretions. However, no such correlate was apparent for immunization with VLP-NP or alum as the adjuvant. Transcriptional profiling of peripheral blood mononuclear cells isolated within the first few hours to days after primary vaccination revealed that NP-adjuvanted vaccines induced a molecular signature similar to that induced by the live attenuated yellow fever viral vaccine. This systems approach identified early blood transcriptional signatures that correlate with Envspecific antibody responses in vaginal secretions and protection against infection. These results demonstrate the adjuvanticity of the NP adjuvant in inducing persistent and protective antibody responses against SIV in RMs with implications for the design of vaccines against human immunodeficiency virus (HIV).

BACKGROUND: Children are susceptible to severe influenza infections and facilitate community transmission. One potential strategy to improve vaccine immunogenicity in children against seasonal influenza involves a trivalent hemagglutinin DNA prime-trivalent inactivated influenza vaccine (IIV3) boost regimen. METHODS: Sites enrolled adolescents, followed by younger children, to receive DNA prime (1 mg or 4 mg) intramuscularly by needle-free jet injector (Biojector), followed by split virus 2012/13 seasonal IIV3 boost by needle and syringe approximately 18 weeks later. A comparator group received IIV3 prime and boost at similar intervals. Primary study objectives included evaluation of the safety and tolerability of the vaccine regimens, with secondary objectives of measuring antibody responses at four weeks post boost by hemagglutination inhibition (HAI) and neutralization assays. RESULTS: Seventy-five children ≥6 to ≤17 years old enrolled. Local reactogenicity was higher after DNA prime compared to IIV3 prime (p<0.001 for pain/tenderness, redness, or swelling), but symptoms were mild to moderate in severity. Systemic reactogenicity was similar between vaccines. Overall, antibody responses were similar among groups, although HAI antibodies revealed a trend towards higher responses following 4 mg DNA-IIV3 compared to IIV3-IIV3. The fold increase of HAI antibodies to A/California/07/2009 [A(H1N1)pdm09] was significantly greater following 4 mg DNA-IIV3 (10.12 fold, 5.60-18.27 95%CI) compared to IIV3-IIV3 (3.86 fold, 2.32-6.44 95%CI). Similar neutralizing titers were observed between regimens, with a trend towards increased response frequencies in 4 mg DNA-IIV3. However, significant differences in fold increase, reported as geometric mean fold ratios, were detected against the H1N1 viruses within the neutralization panel: A/New Caledonia/20/1999 (1.41 fold, 1.10-1.81 95%CI) and A/South Carolina/1/1918 (1.55 fold, 1.27-1.89 95%CI). CONCLUSIONS: In this first pediatric DNA vaccine study conducted in the U.S., the DNA prime-IIV3 boost regimen was safe and well tolerated. In children, the 4 mg DNA-IIV3 regimen resulted in antibody responses comparable to the IIV3-IIV3 regimen.

The search for a preventive vaccine against HIV infection remains an ongoing challenge, indicating the need for novel approaches. Parainfluenza virus 5 (PIV5) is a paramyxovirus replicating in the upper airways that is not associated with any animal or human pathology. In animal models, PIV5-vectored vaccines have shown protection against influenza, RSV, and other human pathogens. Here, we generated PIV5 vaccines expressing HIV envelope (Env) and SIV Gag and administered them intranasally to macaques, followed by boosting with virus-like particles (VLPs) containing trimeric HIV Env. Moreover, we compared the immune responses generated by PIV5-SHIV prime/VLPs boost regimen in naïve vs a control group in which pre-existing immunity to the PIV5 vector was established. We demonstrate for the first time that intranasal administration of PIV5-based HIV vaccines is safe, well-tolerated and immunogenic, and that boosting with adjuvanted trimeric Env VLPs enhances humoral and cellular immune responses. The PIV5 prime/VLPs boost regimen induced robust and durable systemic and mucosal Env-specific antibody titers with functional activities including ADCC and neutralization. This regimen also induced highly polyfunctional antigen-specific T cell responses. Importantly, we show that diminished responses due to PIV5 pre-existing immunity can be overcome in part with VLP protein boosts. Overall, these results establish that PIV5-based HIV vaccine candidates are promising and warrant further investigation including moving on to primate challenge studies.

During the late phase of the Human Immunodeficiency Virus Type-1 (HIV-1) replication cycle, viral Gag proteins and the intact RNA genome are trafficked to specific sub-cellular membranes where virus assembly and budding occurs. Targeting to the plasma membranes of T cells and macrophages is mediated by interactions between the N-terminal matrix (MA) domain of Gag and cellular phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ] molecules. However, in macrophages and dendritic cells, a subset of Gag proteins appears to be targeted to tetraspanin enriched viral compartments, a process that appears to be mediated by MA interactions with the Delta subunit of the cellular Adaptor Protein AP-3 (AP-3δ). We cloned, overexpressed and purified the protein interactive domain of AP-3δ and probed for MA binding by NMR. Unexpectedly, no evidence of binding was observed in these in vitro experiments, even at relatively high protein concentrations (200μM), suggesting that AP-3δ plays an alternative role in HIV-1 assembly.

Neonatal sepsis remains a feared cause of morbidity and mortality in the neonatal period. Maternal, neonatal, and environmental factors are associated with risk of infection, and a combination of prevention strategies, judicious neonatal evaluation, and early initiation of therapy are required to prevent adverse outcomes. This article reviews recent trends in epidemiology and provides an update on risk factors, diagnostic methods, and management of neonatal sepsis.