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Recombinant adeno-associated viral (AAV) vectors are known to safely and efficiently transduce the retina. Among the various AAV serotypes available, AAV2/5 and 2/8 are the most effective for gene transfer to photoreceptors (PR), which are the most relevant targets for gene therapy of inherited retinal degenerations. However, the search for novel AAV serotypes with improved PR transduction is ongoing. In this work we tested vectors derived from five AAV serotypes isolated from porcine tissues (referred to as porcine AAVs, four of which are newly identified) for their ability to transduce both the murine and the cone-enriched pig retina. Porcine AAV vectors expressing EGFP under the control of the CMV promoter were injected subretinally either in C57BL/6 mice or Large White pigs. The resulting retinal tropism was analyzed one month later on histological sections, while levels of PR transduction were assessed by Western blot. Our results show that all porcine AAV transduce murine and porcine retinal pigment epithelium and PR upon subretinal administration. AAV2/po1 and 2/po5 are the most efficient porcine AAVs for murine PR transduction and exhibit the strongest tropism for pig cone PR. The levels of PR transduction obtained with AAV2/po1 and 2/po5 are similar, albeit not superior, to those obtained with AAV2/5 and AAV2/8, which evinces AAV2/po1 and 2/po5 to be promising vectors for retinal gene therapy.

The International Committee on Taxonomy of Viruses (ICTV) organizes the classification of viruses into taxa, but is not responsible for the nomenclature for taxa members. International experts groups, such as the ICTV Study Groups, recommend the classification and naming of viruses and their strains, variants, and isolates. The ICTV Filoviridae Study Group has recently introduced an updated classification and nomenclature for filoviruses. Subsequently, and together with numerous other filovirus experts, a consistent nomenclature for their natural genetic variants and isolates was developed that aims at simplifying the retrieval of sequence data from electronic databases. This is a first important step toward a viral genome annotation standard as sought by the US National Center for Biotechnology Information (NCBI). Here, this work is extended to include filoviruses obtained in the laboratory by artificial selection through passage in laboratory hosts. The previously developed template for natural filovirus genetic variant naming (< virus name > < isolation host-suffix >/< country of sampling >/< year of sampling >/< genetic variant designation >-< isolate designation >) is retained, but it is proposed to adapt the type of information added to each field for laboratory animal-adapted variants. For instance, the full-length designation of an Ebola virus Mayinga variant adapted at the State Research Center for Virology and Biotechnology "Vector" to cause disease in guinea pigs after seven passages would be akin to "Ebola virus VECTOR/C.porcellus-lab/COD/1976/Mayinga-GPA-P7". As was proposed for the names of natural filovirus variants, we suggest using the full-length designation in databases, as well as in the method section of publications. Shortened designations (such as "EBOV VECTOR/C.por/COD/76/May-GPA-P7") and abbreviations (such as "EBOV/May-GPA-P7") could be used in the remainder of the text depending on how critical it is to convey information contained in the full-length name. "EBOV" would suffice if only one EBOV strain/variant/isolate is addressed.

In January 2012, influenza virus researchers from around the world announced a voluntary pause of 60 days on any research involving highly pathogenic avian influenza H5N1 viruses leading to the generation of viruses that are more transmissible in mammals (1). We declared a pause to this important research to provide time to explain the public health benefits of this work, to describe the measures in place to minimize possible risks, and to enable organizations and governments around the world to review their policies (for example, on biosafety, biosecurity, oversight, and communication) regarding these experiments. During the past year, the benefits of this important research have been explained clearly in publications (2-7) and meetings (8-10). Measures to mitigate possible risks of the work have been detailed (11-13). The World Health Organization has released recommendations on laboratory biosafety for those conducting this research (14), and relevant authorities in several countries have reviewed the biosafety, biosecurity, and funding conditions under which further research would be conducted on the laboratory-modified H5N1 viruses (10, 15-17). Thus, acknowledging that the aims of the voluntary moratorium have been met in some countries and are close to being met in others, we declare an end to the voluntary moratorium on avian flu transmission studies. The controversy surrounding H5N1 virus transmission research has highlighted the need for a global approach to dealing with dualuse research of concern. Developing comprehensive solutions to resolve all the issues will take time. Meanwhile, H5N1 viruses continue to evolve in nature. Because H5N1 virus transmission studies are essential for pandemic preparedness and understanding the adaptation of influenza viruses to mammals, researchers who have approval from their governments and institutions to conduct this research safely, under appropriate biosafety and biosecurity conditions, have a public health responsibility to resume this important work. Scientists should not restart their work in countries where, as yet, no decision has been reached on the conditions for H5N1 virus transmission research. At this time, this includes the United States and U.S.-funded research conducted in other countries. Scientists should never conduct this type of research without the appropriate facilities, oversight, and all the necessary approvals. We consider biosafety level 3 conditions with the considerable enhancements (BSL-3+) outlined in the referenced publications (11-13) as appropriate for this type of work, but recognize that some countries may require BSL-4 conditions in accordance with applicable standards (such as Canada). We fully acknowledge that this research—as with any work on infectious agents—is not without risks. However, because the risk exists in nature that an H5N1 virus capable of transmission in mammals may emerge, the benefits of this work outweigh the risks.

The task of international expert groups is to recommend the classification and naming of viruses. The International Committee on Taxonomy of Viruses Filoviridae Study Group and other experts have recently established an almost consistent classification and nomenclature for filoviruses. Here, further guidelines are suggested to include their natural genetic variants. First, this term is defined. Second, a template for full-length virus names (such as “Ebola virus H.sapiens-tc/COD/1995/Kikwit-9510621”) is proposed. These names contain information on the identity of the virus (e.g., Ebola virus), isolation host (e.g., members of the species Homo sapiens), sampling location (e.g., Democratic Republic of the Congo (COD)), sampling year, genetic variant (e.g., Kikwit), and isolate (e.g., 9510621). Suffixes are proposed for individual names that clarify whether a given genetic variant has been characterized based on passage zero material (-wt), has been passaged in tissue/cell culture (-tc), is known from consensus sequence fragments only (-frag), or does (most likely) not exist anymore (-hist). We suggest that these comprehensive names are to be used specifically in the methods section of publications. Suitable abbreviations, also proposed here, could then be used throughout the text, while the full names could be used again in phylograms, tables, or figures if the contained information aids the interpretation of presented data. The proposed system is very similar to the well-known influenzavirus nomenclature and the nomenclature recently proposed for rotaviruses. If applied consistently, it would considerably simplify retrieval of sequence data from electronic databases and be a first important step toward a viral genome annotation standard as sought by the National Center for Biotechnology Information (NCBI). Furthermore, adoption of this nomenclature would increase the general understanding of filovirus-related publications and presentations and improve figures such as phylograms, alignments, and diagrams. Most importantly, it would counter the increasing confusion in genetic variant naming due to the identification of ever more sequences through technological breakthroughs in high-throughput sequencing and environmental sampling.

Top of pageAbstract Severe acute respiratory syndrome (SARS) is a newly described disease caused by SARS-CoV, a novel coronavirus. Due to the limited treatment options and concern over consequences of SARS re-emergence, we have focused on using adenovirus (Ad)-based gene transfer vectors encoding SARS-CoV specific antigens to develop an effective vaccine. We tested the hypothesis that expression of the SARS-CoV spike glycoprotein (S), or its component parts S1 and S2, in E1-E3- Ad serotype 5 vectors can elicit cellular and humoral immune responses against S in immunized mice. A synthetic spike gene was made by an overlapping PCR strategy with codon optimization for mammalian cells. An Ad vector expressing S driven by a CMV promoter was constructed and protein expression was confirmed by Western analysis. To evaluate the immune responses stimulated by this vaccine, 1010 particle units (pu) of the vector was administered by intravenous, intramuscular or subcutaneous routes to C57Bl/6 mice and serum neutralizing antibody titers were measured in an assay assessing the neutralization of SARS-CoV in vitro. Subcutaneous immunization induced anti-SARS-CoV neutralizing antibodies at 2 wk (reciprocal neutralizing titer 32 ± 15) and at 5 wk (690 ± 470) post-administration. Mice immunized by either intramuscular or intravenous routes also developed neutralizing titers (380 ± 96 and 450 ± 78 respectively) by 5 wk post-administration. Immunization by all routes resulted in statistically similar titers (p>0.3 all pairwise comparisons). At a dose of 1011 pu, anti-SARS-CoV neutralizing titers were also measurable (700 ± 160, intravenous; 130 ± 20 intravenous; 450 ± 210 subcutaneous). In contrast, naive mice and mice immunized with AdNull (an Ad with no transgene) had no detectable neutralizing antibody titers at any time point. Cellular immunity stimulated by immunization was evaluated in BALB/c mice injected intravenously with AdnS or AdNull at a dose of 1011 pu. After 2 wk, splenic CD8+ T cells were isolated and exposed to syngeneic target cells stably expressing S. Accumulation of intracellular IFNγ was observed in 8% of CD8+ cells as opposed to 2.5% of cells exposed to syngeneic cells not expressing S. Similar results were observed in C57Bl/6 mice, with 11.5% of CD8+ cells accumulating intracellular IFNγ in response to syngeneic cells expressing S, as opposed to 3.3% of CD8+ cells exposed to syngeneic cell lines not expressing S. The AdNull vaccinated controls did not stimulate antigen-specific IFNγ accumulation in the splenic CD8+ cells. To determine the location of the major cellular epitopes in the spike glycoprotein, Ad vectors and target syngeneic cell lines expressing the N-terminal domain (S1) or the C-terminal domain (S2) of spike were constructed. The specific accumulation of IFNγ in immunized BALB/c mice at 2 wk post-administration was more dependent on the S2 domain than the S1 domain (19% specific intracellular IFNγ accumulation using the S2 domain vs 6.3% with S1). We conclude that immunization with an Ad vaccine vector expressing the SARS-CoV S elicits high titers of SARS-CoV-neutralizing antibodies and that the S2 domain of spike contains the immunodominant CD8+ T-cell epitopes.

Summary The 2013–2016 outbreak of Ebola virus (EBOV) in West Africa was the largest recorded. It began following the cross-species transmission of EBOV from an animal reservoir, most likely bats, into humans, with phylogenetic analysis revealing the co-circulation of several viral lineages. We hypothesized that this prolonged human circulation led to genomic changes that increased viral transmissibility in humans. We generated a synthetic glycoprotein (GP) construct based on the earliest reported isolate and introduced amino acid substitutions that defined viral lineages. Mutant GPs were used to generate a panel of pseudoviruses, which were used to infect different human and bat cell lines. These data revealed that specific amino acid substitutions in the EBOV GP have increased tropism for human cells, while reducing tropism for bat cells. Such increased infectivity may have enhanced the ability of EBOV to transmit among humans and contributed to the wide geographic distribution of some viral lineages., Graphical Abstract, Highlights • EBOV adapted to humans during the West African outbreak • Amino acid substitutions in the EBOV glycoprotein increase human cell tropism • The same glycoprotein amino acid substitutions decrease tropism for bat cells, The Ebola virus acquired amino acid substitutions in its glycoprotein that increased its tropism for human cells during the West African outbreak of 2013–2016.

The recent Ebola outbreak in West Africa is a reminder that correlates of protection against filovirus infection are still not well understood. DNA vectors are a serology-independent platform that allow for vector re-administration with minimal side-effects. We designed a new micro-consensus DNA vaccine that expresses a Zaire Ebolavirus (EBOV) glycoprotein (GP) based on 2002–2008 EBOV outbreak strains. This novel glycoprotein is 3% distant from the GP expressed by the 1976 EBOV Mayinga outbreak strain. The optimized GP DNA vaccine was administered in mice by intramuscular injection followed by electroporation (IM-EP) and elicited strong total IgG antibody and T cell responses. To assess protection from challenge, we administered the GP DNA vaccine or pVax1 control (40ug) to BALB/c mice (n=10/group) by IM-EP at days 0, -7, -14, or -28 before lethal challenge (1000LD50) with a mouse-adapted EBOV Mayinga strain. Importantly we observed 100% protection against the lethal heterologous challenge when the DNA vaccine was administered 28 days before challenge illustrating the potency of this vaccine in this model. More importantly, we observed 100% protection and 90% protection when the DNA vaccine was administered at days -14 and day -7, respectively. Most surprising was the observation of 40% survival in the group that received vaccine at Day 0 (2–3 hours) before challenge. Rapid, short-term protection has been previously observed with a VSV-ZEBOVGP viral vector vaccine but never before with a DNA vaccine. The data suggests that protection in mice following administration of a heterologous DNA vaccine can be afforded by mechanisms independent solely of antibody titers. Ongoing studies are underway in NHPs to investigate these findings.

The recent Ebola outbreak in West Africa has been the deadliest in the history. To prevent future recurrence of such outbreak, better treatments and effective vaccines against Ebola virus are desirable. Among such promising treatments, the Zmapp cocktail containing neutralizing antibodies (13C6, 2G4 and 4G7) has successfully treated some patients. However, the feasibility of using it on large populations especially in developing countries is questionable. To address this potential issue, we propose to employ recombinant vectors derived from adeno-associated virus (rAAV). There are several advantages of using rAAV: because of 1) their safety profile; 2) only one injection (or a few) would be required; 3) the high stability of lyophilized rAAVs at ambient temperature and; 4) the panel of available serotypes. Because of these interesting features, we are currently developing a treatment based on three rAAVs to deliver the genes for the Zmapp cocktail of antibodies. We have already produced at small scale a rAAV expressing the 2G4 antibody. The DNA sequences for the heavy chain and light chains were codon-optimized for better expression in humans and were designed to be expressed from the same gene. A strong promoter (CAG) resistant to silencing in vivo was chosen to drive gene expression of the antibody. The rAAV were produced by transfection using our patented cGMP compatible HEK293 cell line. The production was performed in suspension culture in the absence of serum. Secretion of 2G4 antibody by rAAV transduced cells (HEK293 and CHO cells) was confirmed. The results demonstrated that rAAV-CAG-2G4 was functional and allowed for the correct assembly of the heavy and light chains of 2G4. Purification of 200 mL of rAAV-CAG-2G4 production was performed by ultracentrifugation on an iodixanol density-step gradient. Two other rAAVs coding 13C6 and 4G7 antibodies are in the processed of being constructed and produced in a similar manner. We are also in the process of comparing the efficacy of two serotypes of AAV (9 and DJ) in mice by intranasal delivery. Using the best serotype, the rAAVs will be produced and purified from a starting suspension culture of 20 L. Their efficacy for treating Ebola infections will then be evaluated in a mouse model infected by the virus.

Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.

Karuppiah Muthumani,* Darryl Falzarano,* Emma L. Reuschel, Colleen Tingey, Seleeke Flingai, Daniel O. Villarreal,MeganWise, Ami Patel, Abdullah Izmirly, AbdulelahAljuaid, AleciaM. Seliga, Geoff Soule, Matthew Morrow, Kimberly A. Kraynyak, Amir S. Khan, Dana P. Scott, FriederikeFeldmann,Rachel LaCasse,KimberlyMeade-White,AtsushiOkumura,KennethE.Ugen, Niranjan Y. Sardesai, J. Joseph Kim, Gary Kobinger, Heinz Feldmann, David B. Weiner

There is a critical need to have vaccines that can protect against emerging pandemic influenza viruses. Commonly used influenza vaccines are killed whole virus that protect against homologous and not heterologous virus. Using chickens we have explored the possibility of using live low pathogenic avian influenza (LPAI) A/goose/AB/223/2005 H1N1 or A/WBS/MB/325/2006 H1N2 to induce immunity against heterologous highly pathogenic avian influenza (HPAI) A/chicken/Vietnam/14/2005 H5N1. H1N1 and H1N2 replicated in chickens but did not cause clinical disease. Following infection, chickens developed nucleoprotein and H1 specific antibodies, and reduced H5N1 plaque size in vitro in the absence of H5 neutralizing antibodies at 21 days post infection (DPI). In addition, heterologous cell mediated immunity (CMI) was demonstrated by antigen-specific proliferation and IFN-γ secretion in PBMCs re-stimulated with H5N1 antigen. Following H5N1 challenge of both pre-infected and naive controls chickens housed together, all naive chickens developed acute disease and died while H1N1 or H1N2 pre-infected chickens had reduced clinical disease and 70–80% survived. H1N1 or H1N2 pre-infected chickens were also challenged with H5N1 and naive chickens placed in the same room one day later. All pre-infected birds were protected from H5N1 challenge but shed infectious virus to naive contact chickens. However, disease onset, severity and mortality was reduced and delayed in the naive contacts compared to directly inoculated naive controls. These results indicate that prior infection with LPAI virus can generate heterologous protection against HPAI H5N1 in the absence of specific H5 antibody.

Ebola viruses (EBOV) cause often fatal hemorrhagic fever in several species of simian primates including human. While fruit bats are considered natural reservoir, involvement of other species in EBOV transmission is unclear. In 2009, Reston-EBOV was the first EBOV detected in swine with indicated transmission to humans. In-contact transmission of Zaire-EBOV (ZEBOV) between pigs was demonstrated experimentally. Here we show ZEBOV transmission from pigs to cynomolgus macaques without direct contact. Interestingly, transmission between macaques in similar housing conditions was never observed. Piglets inoculated oro-nasally with ZEBOV were transferred to the room housing macaques in an open inaccessible cage system. All macaques became infected. Infectious virus was detected in oro-nasal swabs of piglets, and in blood, swabs, and tissues of macaques. This is the first report of experimental interspecies virus transmission, with the macaques also used as a human surrogate. Our finding may influence prevention and control measures during EBOV outbreaks.

Ebola virus causes severe hemorrhagic fever in susceptible hosts. Currently, no licensed vaccines or treatments are available; however, several experimental vaccines have been successful in protecting rodents and nonhuman primates (NHPs) from the lethal Zaire ebolavirus (ZEBOV) infection. The objective of this study was to evaluate immune responses correlating with survival in these animals after lethal challenge with ZEBOV. Knockout mice with impaired ability to generate normal T and/or B cell responses were vaccinated and challenged with ZEBOV. Vaccine-induced protection in mice was mainly mediated by B cells and CD4(+) T cells. Vaccinated, outbred guinea pigs and NHPs demonstrated the highest correlation between survival and levels of total immunoglobulin G (IgG) specific to the ZEBOV glycoprotein (ZGP). These results highlight the relevance of total ZGP-specific IgG levels as a meaningful correlate of protection against ZEBOV exposure.

Ebola virus (EBOV) is considered one of the most aggressive infectious agents and is capable of causing death in humans and nonhuman primates (NHPs) within days of exposure. Recent strategies have succeeded in preventing acquisition of infection in NHPs after treatment; however, these strategies are only successful when administered before or minutes after infection. The present work shows that a combination of three neutralizing monoclonal antibodies (mAbs) directed against the Ebola envelope glycoprotein (GP) resulted in complete survival (four of four cynomolgus macaques) with no apparent side effects when three doses were administered 3 days apart beginning at 24 hours after a lethal challenge with EBOV. The same treatment initiated 48 hours after lethal challenge with EBOV resulted in two of four cynomolgus macaques fully recovering. The survivors demonstrated an EBOV-GP-specific humoral and cell-mediated immune response. These data highlight the important role of antibodies to control EBOV replication in vivo, and support the use of mAbs against a severe filovirus infection.

The cytidine deaminase APOBEC3G (A3G) exerts a multifaceted antiviral effect against HIV-1 infection. First, A3G was shown to be able to terminate HIV infection by deaminating the cytosine residues to uracil in the minus strand of the viral DNA during reverse transcription. Also, a number of studies have indicated that A3G inhibits HIV-1 reverse transcription by a non-editing-mediated mechanism. However, the mechanism by which A3G directly disrupts HIV-1 reverse transcription is not fully understood. In the present study, by using a cell-based coimmunoprecipitation (Co-IP) assay, we detected the direct interaction between A3G and HIV-1 reverse transcriptase (RT) in produced viruses and in the cotransfected cells. The data also suggested that their interaction did not require viral genomic RNA bridging or other viral proteins. Additionally, a deletion analysis showed that the RT-binding region in A3G was located between amino acids 65 and 132. Overexpression of the RT-binding polypeptide A3G 65-132 was able to disrupt the interaction between wild-type A3G and RT, which consequently attenuated the anti-HIV effect of A3G on reverse transcription. Overall, this paper provides evidence for the physical and functional interaction between A3G and HIV-1 RT and demonstrates that this interaction plays an important role in the action of A3G against HIV-1 reverse transcription.

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.

UVB-induced cutaneous photodamage/photoaging is characterized by qualitative and quantitative deterioration in dermal extracellular matrix (ECM) components such as collagen and elastic fibers. Disappearance of microfibrillar-associated protein 4 (MFAP-4), a possible limiting factor for cutaneous elasticity, was documented in photoaged dermis, but its function is poorly understood. To characterize its possible contribution to photoprotection, MFAP-4 expression was either augmented or inhibited in a human skin xenograft photodamage murine model and human fibroblasts. Xenografted skin with enhanced MFAP-4 expression was protected from UVB-induced photodamage/photoaging accompanied by the prevention of ECM degradation and aggravated elasticity. Additionally, remarkably increased or decreased fibrillin-1-based microfibril development was observed when fibroblasts were treated with recombinant MFAP-4 or with MFAP-4-specific siRNA, respectively. Immunoprecipitation analysis confirmed direct interaction between MFAP-4 and fibrillin-1. Taken together, our findings reveal the essential role of MFAP-4 in photoprotection and offer new therapeutic opportunities to prevent skin-associated pathologies.

Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against Ebola glycoprotein in mice and guinea pigs. Immunocompetent mice or guinea pigs were given MAbs i.p. in various doses individually or as pools of 3–4 MAbs to test their protection against a lethal challenge with mouse- or guinea pig-adapted EBOV. Each of the 8 MAbs (100 µg) protected mice from a lethal EBOV challenge when administered 1 day before or after challenge. Seven MAbs were effective 2 days post-infection (dpi), with 1 MAb demonstrating partial protection 3 dpi. In the guinea pigs each MAb showed partial protection at 1 dpi, however the mean time to death was significantly prolonged compared to the control group. Moreover, treatment with pools of 3–4 MAbs completely protected the majority of animals, while administration at 2–3 dpi achieved 50–100% protection. This data suggests that the MAbs generated are capable of protecting both animal species against lethal Ebola virus challenge. These results indicate that MAbs particularly when used as an oligoclonal set are a potential therapeutic for post-exposure treatment of EBOV infection., Author Summary Ebola virus (EBOV) causes acute hemorrhagic fever in humans and non-human primates with mortality rates up to 90%. So far there are no effective treatments available. This study evaluates the protective efficacy of 8 monoclonal antibodies (MAbs) against the Ebola virus surface glycoprotein, in mice and guinea pigs. Various combinations and doses of the neutralizing and non-neutralizing MAbs were tested, and a post-exposure treatment protocol was determined. There was 100% survival when guinea pigs received a mix of 3 neutralizing MAbs two days after a challenge with 1,000 LD50 of guinea pig-adapted EBOV. This data suggests that the MAbs generated are effective as a post-exposure therapeutic for a lethal Ebola virus infection. Development of a post-exposure therapeutic for an Ebola virus infection is vital due to the high lethality of the disease, the relative speed in which it kills, and the fact that no vaccine has been approved for human use. Additionally, is it unlikely that preventative vaccines will be employed, because Ebola virus infections occur primarily in Africa, and to date have only killed approximately 2,300 people making it financially unfeasible for a mass vaccination. Therefore, having an effective therapy in the event of an outbreak would be extremely beneficial.

Background Approximately 35% of the North American population and an estimated 90% of the sub-Saharan African population have antibodies against adenovirus serotype 5 (AdHu5) that are capable of neutralizing AdHu5-based vaccines. In mice, intranasal delivery of AdHu5 expressing the Zaire ebolavirus glycoprotein human adenovirus serotype 5 (Ad) containing the genes for the Zaire ebolavirus glycoprotein (ZGP) under the expressional control of a cytomegalovirus immediate early promoter (CMV)) can bypass systemic preexisting immunity, resulting in protection against mouse-adapted Zaire ebolavirus (Mayinga 1976). Methods Guinea pigs administered an adenovirus-based Ebola virus vaccine either intramuscularly or intranasally in the presence of systemically or mucosally induced adenovirus immunity were challenged with a lethal dose of guinea pig-adapted Zaire ebolavirus (Mayinga 1976) (GA-ZEBOV). The humoral immune response was assayed to determine the effect of vaccine delivery route and preexisting immunity. Results Intramuscular or intranasal vaccination fully protected guinea pigs against a lethal GA-ZEBOV challenge. However, intramuscular vaccination in animals with systemically induced preexisting immunity resulted in low survival following challenge. Interestingly, intranasal vaccination protected guinea pigs with systemic preexisting immunity to AdHu5. Mucosal adenoviral immunity induced by intranasal administration of AdHu5 decreased protection following intranasal vaccination with the first-generation but not with the second-generation vaccine. Conclusions Intranasal vaccination is an effective vaccine delivery route in the presence of systemic and, to a lower extent, mucosal preexisting immunity to the vaccine vector in guinea pigs.

Several early pandemic H1N1 influenza isolates cause severe disease in different animals models, while most strains result in mild clinical signs similar to seasonal influenza. In this study, the pathogenesis of the virulent Mexican isolate A/Mexico/InDRE4487/2009 and a mild Canadian isolate A/Canada-AB/RV1532/2009 was compared in ferrets. These viruses differed at nine residues, none of which has been previously identified as virulence factor. The Mexican isolate caused more severe disease and higher mortality, and reached higher peak nasal wash titers. Both viruses grew similarly in the respiratory tract, but only the virulent virus was detected in the gut after day 3. During the acute phase, both strains caused similar lung pathology, however the Mexican isolate induced severe inflammation even after virus clearance. This virus was also associated with a rapid and sustained induction of inflammatory cytokines, indicating that early dysregulation of the host response contributes importantly to the disease outcome.

(See the editorial commentary by Bausch, on pages 179-81.)Reston ebolavirus was recently detected in pigs in the Philippines. Specific antibodies were found in pig farmers, indicating exposure to the virus. This important observation raises the possibility that pigs may be susceptible to Ebola virus infection, including from other species, such as Zaire ebolavirus (ZEBOV), and can transmit to other susceptible hosts.This study investigated whether ZEBOV, a species commonly reemerging in central Africa, can replicate and induce disease in pigs and can be transmitted to naive animals. Domesticated Landrace pigs were challenged through mucosal exposure with a total of 1 ×10(6) plaque-forming units of ZEBOV and monitored for virus replication, shedding, and pathogenesis. Using similar conditions, virus transmission from infected to naive animals was evaluated in a second set of pigs.Following mucosal exposure, pigs replicated ZEBOV to high titers (reaching 10(7) median tissue culture infective doses/mL), mainly in the respiratory tract, and developed severe lung pathology. Shedding from the oronasal mucosa was detected for up to 14 days after infection, and transmission was confirmed in all naive pigs cohabiting with inoculated animals.These results shed light on the susceptibility of pigs to ZEBOV infection and identify an unexpected site of virus amplification and shedding linked to transmission of infectious virus.

DNA-based vaccines, while highly immunogenic in mice, generate significantly weaker responses in primates. Therefore, current efforts are aimed at increasing their immunogenicity, which include optimizing the plasmid/gene, the vaccine formulation and method of delivery. For example, co-immunization with molecular adjuvants encoding an immunomodulatory protein has been shown to improve the antigen (Ag)-specific immune response. Thus, the incorporation of enhancing elements, such as these, may be particularly important in the influenza model in which high titered antibody (Ab) responses are critical for protection. In this regard, we compared the ability of plasmid-encoded high-mobility group box 1 protein (HMGB1), a novel cytokine in which we have previously mutated in order to increase DNA vaccine immunogenicity, with boost Ag-specific immune responses during DNA vaccination with influenza A/PR/8/34 nucleoprotein or the hemagglutinin of A novel H1N1/09. We show that the HMGB1 adjuvant is capable of enhancing adaptive effector and memory immune responses. Although Ag-specific antibodies were detected in all vaccinated animals, a greater neutralizing Ab response was associated with the HMGB1 adjuvant. Furthermore, these responses improved CD8 T+-cell effector and memory responses and provided protection against a lethal mucosal influenza A/PR/8/34 challenge. Thus, co-immunization with HMGB1 has strong in vivo adjuvant activity during the development of immunity against plasmid-encoded Ag.

The identification of immune correlates of protection against highly pathogenic human-adapted influenza is instrumental in the development of the next generation of vaccines. Towards this, ferrets received either one dose of a conventionally produced vaccine, two inoculations of a hemagglutinin (HA)-expressing DNA vaccine, or a prime-boost regimen of the DNA vaccine followed by injection of a HA-expressing adenoviral vector. In addition to the antibody response, ferret-specific interferon-gamma (IFN-γ) ELISpot and flow cytometry assays were developed to follow the cellular immune response. Animals that received the conventional vaccine mounted a humoral response, while the DNA vaccinated groups also developed IFN-γ producing T cells. Upon challenge with the matched highly pathogenic A/South Carolina/1/18 H1N1 influenza A virus, the conventionally vaccinated group developed moderate to severe signs of disease, whereas the DNA vaccinated animals experienced mild disease. In the presence of an antibody response within the protective range, the extent of the T cell response correlated more accurately with reduced morbidity in vaccinated ferrets.

Zaire Ebola virus (ZEBOV) is a pathogen that causes severe hemorrhagic fever in humans and non-human primates. There are currently no licensed vaccines or approved treatments available against ZEBOV infections. The goal of this work was to evaluate different treatment strategies in conjunction with a replication deficient, recombinant human adenovirus serotype 5 (Ad-CAGopt)-based vaccine expressing the Zaire Ebola virus glycoprotein (ZGP) in Ebola infected mice and guinea pigs. Guinea pigs were treated with Ad-CAGoptZGP in combination with different treatment strategies after challenge with guinea pig adapted-ZEBOV (GA-ZEBOV). B10.BR mice were used to further characterize efficacy and immune responses following co-administration of Ad-CAGoptZGP with the most effective treatment: AdHu5 expressing recombinant IFN-α (hereafter termed DEF201) after challenge with a lethal dose of mouse adapted-ZEBOV (MA-ZEBOV). In mice, DEF201 treatment was able to elicit full protection against a lethal dose of MA-ZEBOV when administered 30 minutes after infection. In guinea pigs the Ad-CAGoptZGP and DEF201 combination therapy elicited full protection when treated 30 minutes post-exposure and were a superior treatment to Ad-CAGoptZGP supplemented with recombinant IFN-α protein. Further analysis of the immune response revealed that addition of DEF201 to Ad-CAGoptZGP enhances the resulting adaptive immune response against ZGP. The results highlight the importance of the innate immune response in the prevention of ZEBOV pathogenesis and support further development of the Ad-CAGoptZGP with DEF201 treatment combination for post-exposure therapy against ZEBOV infection.

The administration of what appears to be life-saving serum therapy to two American health volunteers who contracted Ebola in Liberia is a major advance in the treatment of this frequently fatal infection. This advance is a tribute to the scientists who worked collaboratively across borders for

Human adenovirus 5 (AdHu5) vectors are robust vaccine platforms however the presence of naturally-acquired neutralizing antibodies may reduce vector efficacy and potential for re-administration. This study evaluates immune responses and protection following vaccination with a replication-incompetent porcine adenovirus 3 (PAV3) vector as an alternative vaccine to AdHu5 using an avian influenza H5N1 disease model. Vaccine efficacy was evaluated in BALB/c mice following vaccination with different doses of the PAV3 vector expressing an optimized A/Hanoi/30408/2005 H5N1 hemagglutinin antigen (PAV3-HA) and compared with an AdHu5-HA control. PAV3-HA rapidly generated antibody responses, with significant neutralizing antibody titers on day 21, and stronger cellular immune responses detected on day 8, compared to AdHu5-HA. The PAV3-HA vaccine, administered 8 days before challenge, demonstrated improved survival and lower virus load. Evaluation of long-term vaccine efficacy at 12 months post-vaccination showed better protection with the PAV3-HA than with the AdHu5-HA vaccine. Importantly, as opposed to AdHu5, PAV3 vector was not significantly neutralized by human antibodies pooled from over 10,000 individuals. Overall, PAV3-based vector is capable of mediating swift, strong immune responses and offer a promising alternative to AdHu5.

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.

Human immunodeficiency virus type 1 (HIV-1) integrase (IN) is not only a key molecule for HIV genomic integration but also is important in other steps of HIV-1 replication, including reverse transcription, nuclear import, chromatin targeting, virus release and maturation. In this study, we investigated whether expression of the HIV-1 IN C-terminal domain (CTD) polypeptide could affect viral replication. We found that expression of T7 or YFP tagged INcwild type (WT) and mutant INc215, 9AA in virus-producing cells decreased HIV-1 infectivity roughly 3-7 fold in HeLa-?-Gal- CD4/CCR5, CD4+MT4 and C8166 T cells. We further observed that the Pr55gag processing in progeny viruses produced from cells expressing the T7-INcWT or T7-INc215, 9AA was largely inhibited. Results from one-cycle HIV-1 replication revealed that the expression of the IN CTD lead to a moderate inhibition of incoming virus infection by affecting the events prior to integration. By using a lentiviral vector system, we generated a stable CD4+ C8166 T cell line expressing either T7-INcWT or T7-INc215, 9AA and demonstrated that both cell lines became resistant to HIV-1 infection. We conclude that the expression of the HIV-1 IN CTD polypeptide alone can inhibit virus replication by impairing virus maturation and interfering with early step(s) of the viral life cycle.

High antigenic compatibility and low toxicity is associated with xenograft transplantation of porcine tissues in immunodeficient human recipients. We hypothesized that adeno-associated viruses (AAVs) of porcine origin could be highly compatible to human tissues and thus of good efficiency and low toxicity for in vivo gene transfer. Porcine tissues were screened by PCR for the presence of AAV using primers designed to bind conserved regions and amplify variable regions of an alignment of several AAV sequences available on GenBank. We isolated new AAV capsid sequences from porcine tissues and successfully generated a recombinant AAV2/po1 vector by transfection. The AAV2/po1 vector was not cross-neutralized by antisera generated against all other commonly used AAVs (serotype 1, 2, 3, 4, 5, 7 and 8) indicating a distinct antigenic profile. Preexisting immunity to AAVpo1 could not be detected in the human sera evaluated. In mice, AAV2/po1 particles expressing beta-galactosidase or green fluorescent protein demonstrated high transduction efficiency in muscle fibers and the retina after intramuscular or intraocular administration. Biodistribution experiments following systemic administration showed efficient gene transfer exclusively in muscle fibers. Novel AAVs derived from porcine tissues may contribute to the generation of new preventive or curative clinical modalities acceptable for human use.

The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. In this study, we assessed the protective potential of exogenous IFN-α against seasonal and highly pathogenic influenza viruses in ferrets. Intranasal treatment with IFN-α several hours before infection with the H1N1 influenza A virus strain A/USSR/90/77 reduced viral titers in nasal washes at least 100-fold compared to mock-treated controls. IFN-treated animals developed only mild and transient respiratory symptoms, and the characteristic fever peak seen in mock-treated ferrets 2 days after infection was not observed. Repeated application of IFN-α substantially increased the protective effect of the cytokine treatment. IFN-α did not increase survival after infection with the highly pathogenic H5N1 avian influenza A virus strain A/Vietnam/1203/2004. However, viral titers in nasal washes were significantly reduced at days 1 and 3 postinfection. Our study shows that intranasal application of IFN-α can protect ferrets from seasonal influenza viruses, which replicate mainly in the upper respiratory tract, but not from highly pathogenic influenza viruses, which also disseminate to the lung. Based on these results, a more intensive evaluation of IFN-α as an emergency drug against pandemic influenza A is warranted.

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.

Human infections with Ebola virus (EBOV) result in a deadly viral disease known as Ebola hemorrhagic fever. Up to 90% of infected patients die, and there is no available treatment or vaccine. The sporadic human outbreaks are believed to result when EBOV “jumps” from an infected animal to a person and is subsequently transmitted between persons by direct contact with infected blood or body fluids. This study was undertaken to investigate the mechanism by which EBOV can persistently infect and then escape from model cell and animal reservoir systems. We report a model system in which infection of mouse and bat cell lines with EBOV leads to persistence, which can be broken with low levels of lipopolysaccharide or phorbol-12-myristate-13-acetate (PMA). This reactivation depends on the Ras/MAPK pathway through inhibition of RNA-dependent protein kinase and eukaryotic initiation factor 2α phosphorylation and occurs at the level of protein synthesis. EBOV also can be evoked from mice 7 days after infection by PMA treatment, indicating that a similar mechanism occurs in vivo . Our findings suggest that EBOV may persist in nature through subclinical infection of a reservoir species, such as bats, and that appropriate physiological stimulation may result in increased replication and transmission to new hosts. Identification of a presumptive mechanism responsible for EBOV emergence from its reservoir underscores the “hit-and-run” nature of the initiation of human and/or nonhuman primate EBOV outbreaks and may provide insight into possible countermeasures to interfere with transmission.

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.

Background. Mucosal vaccination can offer several advantages over conventional intramuscular immunization to protect against Ebola virus (EBOV) infection, such as immune protection at sites of viral entry into susceptible individuals, and can be administered using needle-free devices. Methods. The present study evaluated oral and nasal vaccination of mice with human adenovirus serotype 5 (Ad) expressing the Zaire ebolavirus glycoprotein (Ad-ZGP) in terms of their protection against and underlying immune responses to EBOV. Results. Similar to intramuscular administration, oral or nasal vaccination of mice with Ad-ZGP fully protected the mice against a lethal challenge with mouse-adapted EBOV. Both T and B cell responses developed in mice receiving oral or nasal vaccination in different body compartments, indicating qualitative improvement of the immune response after mucosal immunization, compared with intramuscular vaccination. Conclusions. Overall, the breadth of the immune response noted after nasal or oral immunization, including stimulation of CD8 + T cells or effector memory T cells from the gastrointestinal tract or the lungs, was superior to that noted after intramuscular administration of the vaccine. The present study showed that adenovirus-based vaccine is effective against EBOV infection in mice after oral and nasal immunization.

The human severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly infectious virus that causes severe respiratory infections in humans. The spike envelope glycoprotein of SARS-CoV, the main determinant of SARS-CoV tropism, was isolated and used to pseudotype a human immunodeficiency virus (HIV)-based vector. Spike-pseudotyped HIV vector was generated and evaluated in vitro on well-differentiated human airway epithelial cells and bronchial explants and in vivo in murine airways. The spike envelope was less efficient at promoting HIV vector transduction of murine airway epithelium than an optimized deletion mutant of the Zaire ebolavirus envelope glycoprotein (NTD6L), which was used as a benchmark. However, spike-pseudotyped HIV vector was substantially more efficient than NTD6L-pseudotyped vector on human airway epithelium as demonstrated by lacZ gene transfer in primary cultures of epithelial cells and bronchial explants. In addition, this study shows that spike-pseudotyped HIV -based vector can efficiently transduce human dendritic cells and epithelial cells of the esophagus, which may have implications in investigating mechanisms of SARS-CoV pathogenesis. Spike-pseudotyped HIV-based vector is a novel lung-directed gene transfer vehicle that holds promise for the treatment of genetic lung diseases such as cystic fibrosis or alpha(1)-antitrypsin deficiency.

Pseudotyping lentiviral vector with other viral surface proteins could be applied for treating genetic anomalies in human skin. In this study, the modification of HIV vector tropism by pseudotyping with the envelope glycoprotein from vesicular stomatitis virus (VSV), the Zaire Ebola (EboZ) virus, murine leukemia virus (MuLV), lymphocytic choriomeningitis virus (LCMV), Rabies or the rabies-related Mokola virus encoding LacZ as a reporter gene was evaluated qualitatively and quantitatively in human skin xenografts. High transgene expression was detected in dermal fibroblasts transduced with VSV-G-, EboZ- or MuLV-pseudotyped HIV vector with tissue irregularities in the dermal compartments following repeated injections of EboZ- or LCMV-pseudotyped vectors. Four weeks after transduction, double-labeling immunofluorescence of beta-galactosidase and involucrin or integrin beta1 demonstrated that VSV-G-, EboZ- or MuLV-pseudotyped HIV vector effectively targeted quiescent epidermal stem cells which underwent terminal differentiation resulting in transgene expression in their progenies. Among the six different pseudotyped HIV-based vectors evaluated, VSV-G-pseudotyped vector was found to be the most efficient viral glycoprotein for cutaneous transduction as demonstrated by the highest level of beta-galactosidase expression and genome copy number evaluated by TaqMan PCR.

First identified in 2012, Middle East respiratory syndrome (MERS) is caused by an emerging human coronavirus, which is distinct from the severe acute respiratory syndrome coronavirus (SARS-CoV), and represents a novel member of the lineage C betacoronoviruses. Since its identification, MERS coronavirus (MERS-CoV) has been linked to more than 1372 infections manifesting with severe morbidity and, often, mortality (about 495 deaths) in the Arabian Peninsula, Europe, and, most recently, the United States. Human-to-human transmission has been documented, with nosocomial transmission appearing to be an important route of infection. The recent increase in cases of MERS in the Middle East coupled with the lack of approved antiviral therapies or vaccines to treat or prevent this infection are causes for concern. We report on the development of a synthetic DNA vaccine against MERS-CoV. An optimized DNA vaccine encoding the MERS spike protein induced potent cellular immunity and antigen-specific neutralizing antibodies in mice, macaques, and camels. Vaccinated rhesus macaques seroconverted rapidly and exhibited high levels of virus-neutralizing activity. Upon MERS viral challenge, all of the monkeys in the control-vaccinated group developed characteristic disease, including pneumonia. Vaccinated macaques were protected and failed to demonstrate any clinical or radiographic signs of pneumonia. These studies demonstrate that a consensus MERS spike protein synthetic DNA vaccine can induce protective responses against viral challenge, indicating that this strategy may have value as a possible vaccine modality against this emerging pathogen.

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.

Importance Safe and effective vaccines and drugs are needed for the prevention and treatment of Ebola virus disease, including following a potentially high-risk exposure such as a needlestick. Objective To assess response to postexposure vaccination in a health care worker who was exposed to the Ebola virus. Design and Setting Case report of a physician who experienced a needlestick while working in an Ebola treatment unit in Sierra Leone on September 26, 2014. Medical evacuation to the United States was rapidly initiated. Given the concern about potentially lethal Ebola virus disease, the patient was offered, and provided his consent for, postexposure vaccination with an experimental vaccine available through an emergency Investigational New Drug application. He was vaccinated on September 28, 2014. Interventions The vaccine used was VSVΔG-ZEBOV, a replicating, attenuated, recombinant vesicular stomatitis virus (serotype Indiana) whose surface glycoprotein gene was replaced by the Zaire Ebola virus glycoprotein gene. This vaccine has entered a clinical trial for the prevention of Ebola in West Africa. Results The vaccine was administered 43 hours after the needlestick occurred. Fever and moderate to severe symptoms developed 12 hours after vaccination and diminished over 3 to 4 days. The real-time reverse transcription polymerase chain reaction results were transiently positive for vesicular stomatitis virus nucleoprotein gene and Ebola virus glycoprotein gene (both included in the vaccine) but consistently negative for Ebola virus nucleoprotein gene (not in the vaccine). Early postvaccination cytokine secretion and T lymphocyte and plasmablast activation were detected. Subsequently, Ebola virus glycoprotein-specific antibodies and T cells became detectable, but antibodies against Ebola viral matrix protein 40 (not in the vaccine) were not detected. Conclusions and Relevance It is unknown if VSVΔG-ZEBOV is safe or effective for postexposure vaccination in humans who have experienced a high-risk occupational exposure to the Ebola virus, such as a needlestick. In this patient, postexposure vaccination with VSVΔG-ZEBOV induced a self-limited febrile syndrome that was associated with transient detection of the recombinant vesicular stomatitis vaccine virus in blood. Strong innate and Ebola-specific adaptive immune responses were detected after vaccination. The clinical syndrome and laboratory evidence were consistent with vaccination response, and no evidence of Ebola virus infection was detected.

A ferret model of severe acute respiratory syndrome (SARS)-CoV infection was used to evaluate the efficacy of an adenovirus vaccine. Animals were subjected to heterologous prime-boost using vectors from human serotype 5 and chimpanzee derived adenoviruses (human AdHu5 and chimpanzee AdC7) expressing spike protein followed by intranasal challenge with SARS-CoV. Vaccination led to a substantial reduction in viral load and prevented the severe pneumonia seen in unvaccinated animals. The same prime-boost strategy was effective in rhesus macaques in eliciting SARS-CoV specific immune responses. These data indicate that a heterologous adenovirus-based prime-boost vaccine strategy could safely stimulate strong immunity that may be needed for complete protection against SARS-CoV infection.

Adenoviral vectors can be used to generate potent humoral and cellular immune responses to transgene products. Use of adenoviral vectors based on non-human isolates may allow for their utilization in populations harbouring neutralizing antibodies to common human serotypes. A vector chimera was constructed using simian adenovirus 22 (a serotype belonging to the species Human adenovirus E) and simian adenovirus 21 (a serotype belonging to the species Human adenovirus B) expressing the Ebola (Zaire) virus glycoprotein (Ad C5/C1-ZGP). This chimeric adenovirus vector was used as a model to test its efficacy as a genetic vaccine and comparisons were made to a vector based on the commonly used human adenovirus C serotype 5 (Adhu5-ZGP). Ebola glycoprotein-specific T- and B-cell responses were measured in B10BR mice vaccinated with either Adhu5-ZGP or Ad C5/C1-ZGP vectors. Both vectors resulted in Ebola glycoprotein-specific gamma interferon-expressing T cells, although the Ad C5/C1-ZGP vector appeared to induce lower frequencies with kinetics slower than those elicited by the Adhu5-ZGP vector. The total immunoglobulin G response to Ebola glycoprotein was similar in sera from mice vaccinated with either vector. Two rhesus macaques vaccinated with the Ad C5/C1-ZGP vector were found to mount T-cell and antibody responses to the Ebola glycoprotein. It was found that a single administration of the chimeric Ad C5/C1-ZGP vector protected mice against a lethal challenge with a mouse-adapted strain of the Ebola (Zaire) virus.

Replication-deficient human adenovirus type 5 (AdH5) vectors can induce strong transgene product-specific cellular and humoral responses. However, many adult humans have neutralizing antibodies (NAbs) against AdH5 as a result of natural infection with this virus. Therefore, a chimpanzee adenovirus C7 (AdC7) vector was developed to circumvent interference by preexisting immunity to AdH5. This study evaluated the impact of preexisting immunity to human adenovirus on the efficacy of adenovirus-based vaccines against the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). Efficacy was assessed after intramuscular injection of the vector into mice and was measured as the frequency of SARS-CoV-specific T cells and NAbs against SARS-CoV. Immunogenicity of the AdH5-based vaccine was significantly attenuated or completely abolished when the preexisting anti-AdH5 NAb titer was higher than 40. Because 27% of human serum samples from the United States tested so far have an anti-AdH5 NAb titer higher than 40, our results suggested that a significant percentage of humans with preexisting anti-AdH5 immunity would not be candidates for vaccination with an AdH5-based genetic vaccine. In contrast, preexisting anti-AdH5 NAbs have a minimal effect on the potency of the AdC7-based genetic vaccine. Taken together, our studies warrant the further development of AdC7 as a vaccine carrier for human trials.

As of 4 July 2013, there have been 134 confirmed cases of human infection and 43 fatalities with a new avian influenza A virus (H7N9) ([1][1]). H7N9 is currently contained in Southeastern China, and the majority of human cases of infection have reported direct exposure to poultry ([2][2]). While no

Emerging infectious diseases pose an important challenge to public health globally. The identification of an etiologic pathogen responsible for an emerging infection in humans can be a major task. The time required to isolate and characterize a new infectious agent sufficiently to adopt control measures and, when possible, to select and make available curative and preventive treatments can be considerable and take years. If the novel pathogen exhibits rapid pathogenesis, causing serious illness and death in a significant percentage of infected individuals, in addition to efficient transmission and spread, serious consequences for the human population can be readily observed. The race between the spread of a high-consequence pathogen and the deployment of adequate public health measures can hardly be satisfactory from a public perspective, because it is a reactive chain of events to the initial spread of the infectious agent. Predicting the emergence of infectious diseases months or years in advance would be a perfect solution but for now has remained a theoretical concept. Improved surveillance systems to detect emerging infectious diseases and new technologies using algorithms specifically developed to isolate and identify causative infectious agents have been emphasized to minimize public health emergencies related to emerging infections. Enhanced surveillance and detection is one of the most significant improvements to regional and global public health of the past several decades. For example, several regional or national networks, such as the Global Public Health Intelligence Network in Canada and the Global Disease Detection program of the Centers for Disease Control and Prevention network in the United States, are informing and cooperating with international networks, such as the Global Outbreak Alert and Response Network developed by the World Health Organization [1–3]. Unfortunately, surveillance and detection methods are region specific, and many regions are currently not being actively monitored. Regions under surveillance oftenmake use of different technologies and approaches that are not yet standardized, thus creating many potential holes in the global network intended to detect new pathogens before they cause damage. More work will be required before these newly developed systems are ideal, but the recognition of their importance and usefulness is a critical step toward enhanced preparedness and response and ultimately prevention. Viruses are responsible for a significant proportion of emerging infectious diseases, the numbers of which may be on an increasing trend overall [4, 5]. It is clear that surveillance and identification of unknown pathogens has greatly improved in the past decade, which has resulted in the concomitant rise in the number of new pathogens being discovered each year. Again, this increase has occurred despite the fact that the detection and identification of previously unknown pathogens currently depends on heterogeneous surveillance systems of variable sensitivity and precision. Regardless, heightened surveillance, detection, and identification of emerging pathogens are currently active and prolific fields of research. Whether there exists a real or apparent increase in the number of viruses emerging in the past 50 years, compared with the number that emerged 50 or 100 years earlier, there is certainly an increasing number of viruses being detected and identified in recent years. A PubMed search using the term “emerging viruses” revealed that the number of scientific articles published on the subject of emerging viruses has jumped from

One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing Escherichia coli beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.

The continuous threat of an influenza pandemic represents one of the biggest challenges in public health. Influenza pandemics are known to be caused by viruses that evolve from animal reservoirs, such as in birds and pigs, and can acquire genetic changes that increase their ability to transmit in humans. Pandemic preparedness plans have been implemented worldwide to mitigate the impact of influenza pandemics. A major obstacle in preventing influenza pandemics is that little is known regarding what makes an influenza virus transmissible in humans. As a consequence, the potential pandemic risk associated with the many different influenza viruses of animals cannot be assessed with any certainty. Recent research breakthroughs identified specific determinants of transmission of H5N1 influenza viruses in ferrets. Responsible research on influenza virus transmission using different animal models is conducted by multiple laboratories in the world using the highest international standards of biosafety and biosecurity practices that effectively prevent the release of transmissible viruses from the laboratory. These standards are regulated and monitored closely by the relevant authorities. This statement is being made by the principal investigators of these laboratories. In two independent studies conducted in two leading influenza laboratories at the University of Wisconsin-Madison and Erasmus MC in Rotterdam, the Netherlands, investigators have proved that viruses possessing a hemagglutinin (HA) protein from highly pathogenic avian H5N1 influenza viruses can become transmissible in ferrets. This is critical information that advances our understanding of influenza transmission. However, more research is needed to determine how influenza viruses in nature become human pandemic threats, so that they can be contained before they acquire the ability to transmit from human to human, or so that appropriate countermeasures can be deployed if adaptation to humans occurs. ![Figure][1] CREDIT: GREG KNOBLOCH/CDC Despite the positive public health benefits these studies sought to provide, a perceived fear that the ferret-transmissible H5 HA viruses may escape from the laboratories has generated intense public debate in the media on the benefits and potential harm of this type of research. We would like to assure the public that these experiments have been conducted with appropriate regulatory oversight in secure containment facilities by highly trained and responsible personnel to minimize any risk of accidental release. Whether the ferret-adapted influenza viruses have the ability to transmit from human to human cannot be tested. We recognize that we and the rest of the scientific community need to clearly explain the benefits of this important research and the measures taken to minimize its possible risks. We propose to do so in an international forum in which the scientific community comes together to discuss and debate these issues. We realize that organizations and governments around the world need time to find the best solutions for opportunities and challenges that stem from the work. To provide time for these discussions, we have agreed on a voluntary pause of 60 days on any research involving highly pathogenic avian influenza H5N1 viruses leading to the generation of viruses that are more transmissible in mammals. In addition, no experiments with live H5N1 or H5 HA reassortant viruses already shown to be transmissible in ferrets will be conducted during this time. We will continue to assess the transmissibility of H5N1 influenza viruses that emerge in nature and pose a continuing threat to human health. [1]: pending:yes