Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.

Without an approved vaccine or treatments, Ebola outbreak management has been limited to palliative care and barrier methods to prevent transmission. These approaches, however, have yet to end the 2014 outbreak of Ebola after its prolonged presence in West Africa. Here we show that a combination of monoclonal antibodies (ZMapp), optimized from two previous antibody cocktails, is able to rescue 100% of rhesus macaques when treatment is initiated up to 5 days post-challenge. High fever, viraemia and abnormalities in blood count and blood chemistry were evident in many animals before ZMapp intervention. Advanced disease, as indicated by elevated liver enzymes, mucosal haemorrhages and generalized petechia could be reversed, leading to full recovery. ELISA and neutralizing antibody assays indicate that ZMapp is cross-reactive with the Guinean variant of Ebola. ZMapp exceeds the efficacy of any other therapeutics described so far, and results warrant further development of this cocktail for clinical use.

Ebola virus transmission in guinea pigs.

UNLABELLED: Ebola virus (EBOV) transmission is currently poorly characterized and is thought to occur primarily by direct contact with infectious material; however transmission from swine to nonhuman primates via the respiratory tract has been documented. To establish an EBOV transmission model for performing studies with statistical significance, groups of six guinea pigs (gps) were challenged intranasally (i.n.) or intraperitoneally (i.p.) with 10,000 times the 50% lethal dose (LD50) of gp-adapted EBOV, and naive gps were then introduced as cage mates for contact exposure at 1 day postinfection (p.i.). The animals were monitored for survival and clinical signs of disease and quantitated for virus shedding postexposure. Changes in the duration of contact of naive gps with infected animals were evaluated for their impact on transmission efficiency. Transmission was more efficient from i.n.- than from i.p.-challenged gps, with 17% versus 83% of naive gps surviving exposure, respectively. Virus shedding was detected beginning at 3 days p.i. from both i.n.- and i.p.-challenged animals. Contact duration positively correlated with transmission efficiency, and the abrogation of direct contact between infected and naive animals through the erection of a steel mesh was effective at stopping virus spread, provided that infectious animal bedding was absent from the cages. Histopathological and immunohistochemical findings show that i.n.-infected gps display enhanced lung pathology and EBOV antigen in the trachea, which supports increased virus transmission from these animals. The results suggest that i.n.-challenged gps are more infectious to naive animals than their systemically infected counterparts and that transmission occurs through direct contact with infectious materials, including those transported through air movement over short distances. IMPORTANCE: Ebola is generally thought to be spread between humans though infectious bodily fluids. However, a study has shown that Ebola can be spread from pigs to monkeys without direct contact. Further studies have been hampered, because an economical animal model for Ebola transmission is not available. To address this, we established a transmission model in guinea pigs and determined the mechanisms behind virus spread. The survival data, in addition to microscopic examination of lung and trachea sections, show that mucosal infection of guinea pigs is an efficient model for Ebola transmission. Virus spread is increased with longer contact times with an infected animal and is possible without direct contact between an infected and a naive host but can be stopped if infectious materials are absent. These results warrant consideration for the development of future strategies against Ebola transmission and for a better understanding of the parameters involved in virus spread.

Optimization of Prime-Boost Vaccination Strategies Against Mouse-Adapted Ebolavirus in a Short-Term Protection Study.

In nonhuman primates, complete protection against an Ebola virus (EBOV) challenge has previously been achieved after a single injection with several vaccine platforms. However, long-term protection against EBOV after a single immunization has not been demonstrated to this date. Interestingly, prime-boost regimens have demonstrated longer protection against EBOV challenge, compared with single immunizations. Since prime-boost regimens have the potential to achieve long-term protection, determining optimal vector combinations is crucial. However, testing prime-boost efficiency in long-term protection studies is time consuming and resource demanding. Here, we investigated the optimal prime-boost combination, using DNA, porcine-derived adeno-associated virus serotype 6 (AAV-po6), and human adenovirus serotype 5 (Ad5) vector, in a short-term protection study in the mouse model of EBOV infection. In addition, we also investigated which immune parameters were indicative of a strong boost. Each vaccine platform was titrated in mice to identify which dose (single immunization) induced approximately 20% protection after challenge with a mouse-adapted EBOV. These doses were then used to determine the protection efficacy of various prime-boost combinations, using the same mouse model. In addition, humoral and cellular immune responses against EBOV glycoprotein were analyzed by an enzyme-linked immunosorbent assay, a neutralizing antibody assay, and an interferon γ-specific enzyme-linked immunospot assay. When DNA was used as a prime, Ad5 boost induced the best protection, which correlated with a higher cellular response. In contrast, when AAV-po6 or Ad5 were injected first, better protection was achieved after DNA boost, and this correlated with a higher total glycoprotein-specific immunoglobulin G titer. Prime-boost regimens using independent vaccine platforms may provide a useful strategy to induce long-term immune protection against filoviruses.

Induction of broad cytotoxic T cells by protective DNA vaccination against Marburg and Ebola.

Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported. We developed a synthetic polyvalent-filovirus DNA vaccine against Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV). Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, whereas murine T-cell responses were extensively analyzed using a novel modified assay described herein. Vaccination was highly potent, elicited robust neutralizing antibodies, and completely protected against MARV and ZEBOV challenge. Comprehensive T-cell analysis revealed cytotoxic T lymphocytes (CTLs) of great magnitude, epitopic breadth, and Th1-type marker expression. This model provides an important preclinical tool for studying protective immune correlates that could be applied to existing platforms. Data herein support further evaluation of this enhanced gene-based approach in nonhuman primate studies for in depth analyses of T-cell epitopes in understanding protective efficacy.

Novel adeno-associated viruses derived from pig tissues transduce most major organs in mice.

Recently, development of Adeno-associated virus (AAV) vectors has been focusing on expanding the genetic diversity of vectors from existing sequences via directed evolution or epitope remapping. Apart from intelligent design, AAV isolation from natural sources remains an important source of new AAVs with unique biological features. In this study, several new AAV sequences were isolated from porcine tissues (AAVpo2.1, -po4, -po5, and -po6), which aligned in divergent new clades. Viral particles generated from these sequences displayed tissue tropism and transduction efficiency profile specific to each porcine-derived AAV. When delivered systemically, AAVpo2.1 targeted the heart, kidney, and muscle, AAVpo5 performed poorly but was able to transduce muscle fibers when injected intramuscularly, whereas AAVpo4 and -po6 efficiently transduced all the major organs sampled, contending with 'gold-standard' AAVs. When delivered systemically, AAVpo4 and -po6 were detected by polymerase chain reaction (PCR) and histochemical staining of the transgene product in adult mouse brain, suggesting that these vectors can pass through the blood-brain barrier with efficiencies that may be useful for the development of therapeutic approaches. Porcine tissues are antigenically similar to human tissues and by inference, porcine AAVs may provide fresh tools to contribute to the development of gene therapy-based solutions to human diseases.