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Crimean-Congo hemorrhagic fever virus (CCHFV, order Bunyavirales, family Nairoviridae, genus Orthonairovirus) is the tick-borne etiological agent of Crimean-Congo hemorrhagic fever (CCHF) in humans. Animals are generally susceptible to CCHFV infection but refractory to disease. Small animal models are limited to interferon-deficient mice, that develop acute fatal disease following infection. Here, using a ZsGreen1- (ZsG) expressing reporter virus (CCHFV/ZsG), we examine tissue tropism and dissemination of virus in interferon-α/β receptor knock-out (Ifnar-/-) mice. We demonstrate that CCHFV/ZsG retains in vivo pathogenicity comparable to wild-type virus. Interestingly, despite high levels of viral RNA in all organs assessed, 2 distribution patterns of infection were observed by both fluorescence and immunohistochemistry (IHC), corresponding to the permissiveness of organ tissues. To further investigate viral dissemination and to temporally define cellular targets of CCHFV in vivo, mice were serially euthanized at different stages of disease. Flow cytometry was used to characterize CCHFV-associated alterations in hematopoietic cell populations and to classify infected cells in the blood, lymph node, spleen, and liver. ZsG signal indicated that mononuclear phagocytic cells in the lymphatic tissues were early targets of infection; in late-stage infection, overall, the highest levels of signal were detected in the liver, and ZsG was found in both antigen-presenting and lymphocyte cell populations., Author summary Human infection by tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV) can result in severe disease with up to 30% case fatality rates. While CCHFV is known to be hepatotropic, the presence and implications of virus in other tissues are less clear. Furthermore, to date, early cellular targets of infection in a CCHFV disease model have not been investigated in detail. Here, using a recombinant reporter CCHFV expressing the fluorescent protein ZsGreen1 (ZsG; CCHFV/ZsG) in interferon-α/β receptor knock-out (Ifnar-/-) mice, which develop acute fatal disease following infection, we investigate both cellular and tissue targets of infection. Importantly, we find that CCHFV/ZsG infection demonstrated comparable pathogenicity to wild-type virus in Ifnar-/- mice. We used in situ visualization of fluorescent signal in tissues to assess viral dissemination throughout the course of infection, and found robust viral signal in reproductive tissues, previously unrecognized as sites of CCHFV infection. We also used flow cytometry to detect intracellular fluorescent signal, and identified initial target cells of CCHFV infection as macrophage and monocyte populations in lymphatic tissues. These findings support a central role of immune cells in early virus dissemination, and a need for further investigations into reproductive tract involvement in human CCHFV infection.

The development of an effective vaccine against HIV infection remains a global priority. Dendritic cell (DC)-based HIV immunotherapeutic vaccine is a promising approach which aims at optimizing the HIV-specific immune response using primed DCs to promote and enhance both the cellular and humoral arms of immunity. Since the Ebola virus envelope glycoprotein (EboGP) has strong DC-targeting ability, we investigated whether EboGP is able to direct HIV particles towards DCs efficiently and promote potent HIV-specific immune responses. Our results indicate that the incorporation of EboGP into non-replicating virus-like particles (VLPs) enhances their ability to target human monocyte-derived dendritic cells (MDDCs) and monocyte-derived macrophages (MDMs). Also, a mucin-like domain deleted EboGP (EboGPΔM) can further enhanced the MDDCs and MDMs-targeting ability. Furthermore, we investigated the effect of EboGP on HIV immunogenicity in mice, and the results revealed a significantly stronger HIV-specific humoral immune response when immunized with EboGP-pseudotyped HIV VLPs compared with those immunized with HIV VLPs. Splenocytes harvested from mice immunized with EboGP-pseudotyped HIV VLPs secreted increased levels of macrophage inflammatory proteins-1α (MIP-1α) and IL-4 upon stimulation with HIV Env and/or Gag peptides compared with those harvested from mice immunized with HIV VLPs. Collectively, this study provides evidence for the first time that the incorporation of EboGP in HIV VLPs can facilitate DC and macrophage targeting and induce more potent immune responses against HIV.

An unprecedented Ebola virus (EBOV) epidemic occurred in 2013–2016 in West Africa. Over this time the epidemic exponentially grew and moved to Europe and North America, with several imported cases and many Health Care Workers (HCW) infected. Better understanding of EBOV infection patterns in different body compartments is mandatory to develop new countermeasures, as well as to fully comprehend the pathways of human-to-human transmission. We have longitudinally explored the persistence of EBOV-specific negative sense genomic RNA (neg-RNA) and the presence of positive sense RNA (pos-RNA), including both replication intermediate (antigenomic-RNA) and messenger RNA (mRNA) molecules, in the upper and lower respiratory tract, as compared to plasma, in a HCW infected with EBOV in Sierra Leone, who was hospitalized in the high isolation facility of the National Institute for Infectious Diseases “Lazzaro Spallanzani” (INMI), Rome, Italy. We observed persistence of pos-RNA and neg-RNAs in longitudinally collected specimens of the lower respiratory tract, even after viral clearance from plasma, suggesting possible local replication. The purpose of the present study is to enhance the knowledge on the biological features of EBOV that can contribute to the human-to-human transmissibility and to develop effective intervention strategies. However, further investigation is needed in order to better understand the clinical meaning of viral replication and shedding in the respiratory tract., Author Summary An unprecedented Ebola outbreak occurred in 2013–2016 in West Africa. In order to better understand EBOV infection patterns in different body compartments, we have longitudinally explored the presence of already assessed markers of ongoing EBOV replication (negative sense genomic RNA and positive sense RNA) in the upper and lower respiratory tract, as compared to plasma and other body compartments, in a Health Care Worker infected with EBOV in Sierra Leone, who was hospitalized in the high isolation facility of the National Institute for Infectious Diseases “Lazzaro Spallanzani” (INMI), Rome, Italy. The presence of total EBOV RNA and replication markers was observed in specimens of the lower respiratory tract, even after viral clearance from plasma, suggesting possible local replication. Our results contribute to the knowledge on the biological features of EBOV and shed light on the potential role of respiratory compartment in human-to-human transmissibility.

The past year has marked the most devastating Ebola outbreak the world has ever witnessed, with over 28,000 cases and over 11,000 deaths. Ebola virus (EBOV) has now been around for almost 50 years. In this review, we discuss past and present outbreaks of EBOV and how those variants evolved over time. We explore and discuss selective pressures that drive the evolution of different Ebola variants, and how they may modify the efficacy of therapeutic treatments and vaccines currently being developed. Finally, given the unprecedented size and spread of the outbreak, as well as the extended period of replication in human hosts, specific attention is given to the 2014-2015 West African outbreak variant (Makona).

Ebola viruses (EBOV) are filamentous single-stranded RNA viruses of the family Filoviridae. Zaire ebolavirus (ZEBOV) causes severe haemorrhagic fever in humans, great apes and non-human primates (NHPs) with high fatality rates. In contrast, Reston ebolavirus (REBOV), the only species found outside Africa, is lethal to some NHPs but has never been linked to clinical disease in humans despite documented exposure. REBOV was isolated from pigs in the Philippines and subsequent experiments confirmed the susceptibility of pigs to both REBOV and ZEBOV with predilection for the lungs. However, only ZEBOV caused severe lung pathology in 5-6 weeks old pigs leading to respiratory distress. To further elucidate the mechanisms for lung pathology, microarray analysis of changes in gene expression was performed on lung tissue from ZEBOV-infected pigs. Furthermore, systemic effects were monitored by looking at changes in peripheral blood leukocyte subsets and systemic cytokine responses. Following oro-nasal challenge, ZEBOV replicated mainly in the respiratory tract, causing severe inflammation of the lungs and consequently rapid and difficult breathing. Neutrophils and macrophages infiltrated the lungs but only the latter were positive for ZEBOV antigen. Genes for proinflammatory cytokines, chemokines and acute phase proteins, known to attract immune cells to sites of infection, were upregulated in the lungs, causing the heavy influx of cells into this site. Systemic effects included a decline in the proportion of monocyte/dendritic and B cells and a mild proinflammatory cytokine response. Serum IgM was detected on day 5 and 6 post infection. In conclusion, a dysregulation/over-activation of the pulmonary proinflammatory response may play a crucial role in the pathogenesis of ZEBOV infection in 5-6 weeks old pigs by attracting inflammatory cells to the lungs.

Human skin color is predominantly determined by melanin produced in melanosomes within melanocytes and subsequently distributed to keratinocytes. There are many studies that have proposed mechanisms underlying ethnic skin color variations, whereas the processes involved from melanin synthesis in melanocytes to the transfer of melanosomes to keratinocytes are common among humans. Apart from the activities in the melanogenic rate-limiting enzyme, tyrosinase, in melanocytes and the amounts and distribution patterns of melanosomes in keratinocytes, the abilities of the actin-associated factors in charge of melanosome transport within melanocytes also regulate pigmentation. Mutations in genes encoding melanosome transport-related molecules, such as MYO5A, RAB27A and SLAC-2A, have been reported to cause a human pigmentary disease known as Griscelli syndrome, which is associated with diluted skin and hair color. Thus we hypothesized that process might play a role in modulating skin color variations. To address that hypothesis, the correlations of expression of RAB27A and its specific effector, SLAC2-A, to melanogenic ability were evaluated in comparison with tyrosinase, using human melanocytes derived from 19 individuals of varying skin types. Following the finding of the highest correlation in RAB27A expression to the melanogenic ability, darkly-pigmented melanocytes with significantly higher RAB27A expression were found to transfer significantly more melanosomes to keratinocytes than lightly-pigmented melanocytes in co-culture and in human skin substitutes (HSSs) in vivo, resulting in darker skin color in concert with the difference observed in African-descent and Caucasian skins. Additionally, RAB27A knockdown by a lentivirus-derived shRNA in melanocytes concomitantly demonstrated a significantly reduced number of transferred melanosomes to keratinocytes in co-culture and a significantly diminished epidermal melanin content skin color intensity (ΔL* = 4.4) in the HSSs. These data reveal the intrinsically essential role of RAB27A in human ethnic skin color determination and provide new insights for the fundamental understanding of regulatory mechanisms underlying skin pigmentation.

The neuraminidase inhibitor oseltamivir is currently used for treatment of patients infected with the pandemic A/H1N1 (pH1N1) influenza virus, although drug-resistant mutants can emerge rapidly and possibly be transmitted. We describe the characteristics of a pair of oseltamivir-resistant and oseltamivir-susceptible pH1N1 clinical isolates that differed by a single change (H274Y) in the neuraminidase protein. Viral fitness of pH1N1 isolates was assessed in vitro by determining replication kinetics in MDCK α2,6 cells and in vivo by performing experimental infections of BALB/c mice and ferrets. Despite slightly reduced propagation of the mutant isolate in vitro during the first 24 h, the wild-type (WT) and mutant resistant viruses induced similar maximum weight loss in mice and ferrets with an identical pyrexic response in ferrets (AUC of 233.9 and 233.2, P = 0.5156). Similarly, comparable titers were obtained for the WT and the mutant strains on days 1, 3, 6 and 9 post-infection in mouse lungs and on days 1–7 in ferret nasal washes. A more important perivascular (day 6) and pleural (days 6 and 12) inflammation was noted in the lungs of mice infected with the H274Y mutant, which correlated with increased pulmonary levels of IL-6 and KC. Such increased levels of IL-6 were also observed in lymph nodes of ferrets infected with the mutant strain. Furthermore, the H274Y mutant strain was transmitted to ferrets. In conclusion, viral fitness of the H274Y pH1N1 isolate is not substantially altered and has the potential to induce severe disease and to disseminate., Author Summary During the 2009 pandemic of the novel A/H1N1 (pH1N1) virus, the World Health Organization recommended oseltamivir as first-line agent for treatment of patients with severe infections leading to hospitalization and for those with underlying diseases predisposing to pulmonary complications. Oseltamivir-resistant isolates started to emerge at the end of June 2009 with now more than 100 strains reported worldwide including a few outbreaks where transmission of resistant viruses may have occurred. We characterized the fitness of a pair of oseltamivir-susceptible and oseltamivir-resistant strains emerging from the same familial cluster and that differed by only a single change (H274Y) in the neuraminidase protein. We found that the drug-resistant (mutant) virus was at least as virulent as the drug-susceptible (wild-type) virus in mice and ferrets. Based on these data, we believe that the H274Y pH1N1 mutant strain has the potential to disseminate in the population and to eventually replace the susceptible strain, a phenomenon that has been already observed with seasonal A/Brisbane/59/2007-like (H1N1) viruses.

Background: The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in postexposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP). The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP) and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector. Methodology/Principal Findings: Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (AdCAGoptZGP). Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge. Conclusions/Significance: We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the previous generation adenovirus-based Ebola vaccine. Understanding and improving the molecular components of adenovirus-based vaccines can produce potent, optimized product, useful for vaccination and post-exposure therapy.

Pre-existing immunity to human adenovirus serotype 5 (Ad5) is common in the general population. Bypassing pre-existing immunity could maximize Ad5 vaccine efficacy. Vaccination by the intramuscular (I.M.), nasal (I.N.) or oral (P.O.) route with Ad5 expressing Ebola Zaire glycoprotein (Ad5-ZGP) fully protected naïve mice against lethal challenge with Ebola. In the presence of pre-existing immunity, only mice vaccinated I.N. survived. The frequency of IFN-gamma+ CD8+ T cells was reduced by 80% and by 15% in animals vaccinated by the I.M. and P.O. routes respectively. Neutralizing antibodies could not be detected in serum from either treatment group. Pre-existing immunity did not compromise the frequency of IFN-gamma+ CD8+ T cells (3.9+/-1% naïve vs. 3.6+/-1% pre-existing immunity, PEI) nor anti-Ebola neutralizing antibody (NAB, 40+/-10 reciprocal dilution, both groups). The number of INF-gamma+ CD8+ cells detected in bronchioalveolar lavage fluid (BAL) after I.N. immunization was not compromised by pre-existing immunity to Ad5 (146+/-14, naïve vs. 120+/-16 SFC/million MNCs, PEI). However, pre-existing immunity reduced NAB levels in BAL by approximately 25% in this group. To improve the immune response after oral vaccination, the Ad5-based vaccine was PEGylated. Mice given the modified vaccine did not survive challenge and had reduced levels of IFN-gamma+ CD8+ T cells 10 days after administration (0.3+/-0.3% PEG vs. 1.7+/-0.5% unmodified). PEGylation did increase NAB levels 2-fold. These results provide some insight about the degree of T and B cell mediated immunity necessary for protection against Ebola virus and suggest that modification of the virus capsid can influence the type of immune response elicited by an Ad5-based vaccine.

Background The persistent evolution of highly pathogenic avian influenza (HPAI) highlights the need for novel vaccination techniques that can quickly and effectively respond to emerging viral threats. We evaluated the use of optimized consensus influenza antigens to provide broad protection against divergent strains of H5N1 influenza in three animal models of mice, ferrets, and non-human primates. We also evaluated the use of in vivo electroporation to deliver these vaccines to overcome the immunogenicity barrier encountered in larger animal models of vaccination. Methods and Findings Mice, ferrets and non-human primates were immunized with consensus plasmids expressing H5 hemagglutinin (pH5HA), N1 neuraminidase (pN1NA), and nucleoprotein antigen (pNP). Dramatic IFN-γ-based cellular immune responses to both H5 and NP, largely dependent upon CD8+ T cells were seen in mice. Hemaggutination inhibition titers classically associated with protection (>1:40) were seen in all species. Responses in both ferrets and macaques demonstrate the ability of synthetic consensus antigens to induce antibodies capable of inhibiting divergent strains of the H5N1 subtype, and studies in the mouse and ferret demonstrate the ability of synthetic consensus vaccines to induce protection even in the absence of such neutralizing antibodies. After challenge, protection from morbidity and mortality was seen in mice and ferrets, with significant reductions in viral shedding and disease progression seen in vaccinated animals. Conclusions By combining several consensus influenza antigens with in vivo electroporation, we demonstrate that these antigens induce both protective cellular and humoral immune responses in mice, ferrets and non-human primates. We also demonstrate the ability of these antigens to protect from both morbidity and mortality in a ferret model of HPAI, in both the presence and absence of neutralizing antibody, which will be critical in responding to the antigenic drift that will likely occur before these viruses cross the species barrier to humans.

The close immunological and physiological resemblance with humans makes non-human primates a valuable model for studying influenza virus pathogenesis and immunity and vaccine efficacy against infection. Although both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of susceptibility to infection and disease has not yet been performed. In the current study a head-to-head comparison was made between these species, by using a recently described swine-origin pandemic H1N1 strain, A/Mexico/InDRE4487/2009. In comparison to rhesus macaques, cynomolgus macaques developed significantly higher levels of virus replication in the upper airways and in the lungs, involving both peak level and duration of virus production, as well as higher increases in body temperature. In contrast, clinical symptoms, including respiratory distress, were more easily observed in rhesus macaques. Expression of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, was 50 to 73 times more abundant in trachea and bronchus of cynomolgus macaques relative to rhesus macaques. The study also shows that common marmosets, a New World non-human primate species, are susceptible to infection with pandemic H1N1. The study results favor the cynomolgus macaque as model for pandemic H1N1 influenza virus research because of the more uniform and high levels of virus replication, as well as temperature increases, which may be due to a more abundant expression of the main human influenza virus receptor in the trachea and bronchi.

Certain short peptides do not occur in humans and are rare or non-existent in the universal proteome. Antigens that contain rare amino acid sequences are in general highly immunogenic and may activate different arms of the immune system. We first generated a list of rare, semi-common, and common 5-mer peptides using bioinformatics tools to analyze the UniProtKB database. Experimental observations indicated that rare and semi-common 5-mers generated stronger cellular responses in comparison with common-occurring sequences. We hypothesized that the biological process responsible for this enhanced immunogenicity could be used to positively modulate immune responses with potential application for vaccine development. Initially, twelve rare 5-mers, 9-mers, and 13-mers were incorporated in frame at the end of an H5N1 hemagglutinin (HA) antigen and expressed from a DNA vaccine. The presence of some 5-mer peptides induced improved immune responses. Adding one 5-mer peptide exogenously also offered improved clinical outcome and/or survival against a lethal H5N1 or H1N1 influenza virus challenge in BALB/c mice and ferrets, respectively. Interestingly, enhanced anti-HBsAg antibody production by up to 25-fold in combination with a commercial Hepatitis B vaccine (Engerix-B, GSK) was also observed in BALB/c mice. Mechanistically, NK cell activation and dependency was observed with enhancing peptides ex vivo and in NK-depleted mice. Overall, the data suggest that rare or non-existent oligopeptides can be developed as immunomodulators and supports the further evaluation of some 5-mer peptides as potential vaccine adjuvants.