Natural History of Aerosol Exposure with Marburg Virus in Rhesus Macaques

Marburg virus causes severe and often lethal viral disease in humans, and there are currently no Food and Drug Administration (FDA) approved medical countermeasures. The sporadic occurrence of Marburg outbreaks does not allow for evaluation of countermeasures in humans, so therapeutic and vaccine candidates can only be approved through the FDA animal rule—a mechanism requiring well-characterized animal models in which efficacy would be evaluated. Here, we describe a natural history study where rhesus macaques were surgically implanted with telemetry devices and central venous catheters prior to aerosol exposure with Marburg-Angola virus, enabling continuous physiologic monitoring and blood sampling without anesthesia. After a three to four day incubation period, all animals developed fever, viremia, and lymphopenia before developing tachycardia, tachypnea, elevated liver enzymes, decreased liver function, azotemia, elevated D-dimer levels and elevated pro-inflammatory cytokines suggesting a systemic inflammatory response with organ failure. The final, terminal period began with the onset of sustained hypotension, dehydration progressed with signs of major organ hypoperfusion (hyperlactatemia, acute kidney injury, hypothermia), and ended with euthanasia or death. The most significant pathologic findings were marked infection of the respiratory lymphoid tissue with destruction of the tracheobronchial and mediastinal lymph nodes, and severe diffuse infection in the liver, and splenitis

Detailed analysis of the African green monkey model of Nipah virus disease.

Henipaviruses are implicated in severe and frequently fatal pneumonia and encephalitis in humans. There are no approved vaccines or treatments available for human use, and testing of candidates requires the use of well-characterized animal models that mimic human disease. We performed a comprehensive and statistically-powered evaluation of the African green monkey model to define parameters critical to disease progression and the extent to which they correlate with human disease. African green monkeys were inoculated by the intratracheal route with 2.5 × 10(4) plaque forming units of the Malaysia strain of Nipah virus. Physiological data captured using telemetry implants and assessed in conjunction with clinical pathology were consistent with shock, and histopathology confirmed widespread tissue involvement associated with systemic vasculitis in animals that succumbed to acute disease. In addition, relapse encephalitis was identified in 100% of animals that survived beyond the acute disease phase. Our data suggest that disease progression in the African green monkey is comparable to the variable outcome of Nipah virus infection in humans

DNA vaccines elicit durable protective immunity against individual or simultaneous infections with Lassa and Ebola viruses in guinea pigs

We previously developed optimized DNA vaccines against both Lassa fever and Ebola hemorrhagic fever viruses and demonstrated that they were protective individually in guinea pig and nonhuman primate models. In this study, we vaccinated groups of strain 13 guinea pigs two times, four weeks apart with 50 µg of each DNA vaccine or a mock vaccine at discrete sites by intradermal electroporation. Five weeks following the second vaccinations, guinea pigs were exposed to lethal doses of Lassa virus, Ebola virus, or a combination of both viruses simultaneously. None of the vaccinated guinea pigs, regardless of challenge virus and including the coinfected group, displayed weight loss, fever or other disease signs, and all survived to the study endpoint. All of the mock-vaccinated guinea pigs that were infected with Lassa virus, and all but one of the EBOV-infected mock-vaccinated guinea pigs succumbed. In order to determine if the dual-agent vaccination strategy could protect against both viruses if exposures were temporally separated, we held the surviving vaccinates in BSL-4 for approximately 120 days to perform a cross-challenge experiment in which guinea pigs originally infected with Lassa virus received a lethal dose of Ebola virus and those originally infected with Ebola virus were infected with a lethal dose of Lassa virus. All guinea pigs remained healthy and survived to the study endpoint. This study clearly demonstrates that DNA vaccines against Lassa and Ebola viruses can elicit protective immunity against both individual virus exposures as well as in a mixed-infection environment

Viral RNA in PBMC and throat, nasal, and rectal swabs as measured by qRT-PCR.

<p>NiV-specific qRT-PCR was performed on RNA extracted from PBMC samples and throat, nasal, and rectal swab clarified homogenates. The results for PBMC samples are shown in (A); black lines and symbols represent animals that succumbed, and grey lines and symbols represent animals that survived to end of study. The results for throat (black bars), nasal (grey bars), and rectal (white bars with black outline) swabs are shown in (B)-(E).</p

Immunohistochemistry.

<p>1 = 1–10 cells/high power field (hpf).</p><p>2 = 11–20 cells/hpf.</p><p>3 = 21–40 cells/hpf.</p><p>4 = >40 cells/hpf.</p><p>D = Diffuse.</p><p>F = Focal.</p><p>M = Multifocal.</p><p>+ = intense staining.</p><p>ND = Not detected.</p><p>Blush = staining was light and non-specific.</p><p>Immunohistochemistry.</p

Respiratory rate as measured by telemetry.

<p>ITS telemetry devices were used to collect respiratory rate data. (A) NHP 1; (B) NHP 2; (C) NHP 4. The grey line represents baseline (mean of the telemetry data collected in the six days prior to challenge) for each animal. The diamonds represent statistically significant respiratory rate values (> +3 SD [♦] above or > -3 SD [♦] below baseline) or non-significant values (♦). bpm = beats per minute.</p

Heart rate as measured by telemetry.

<p>ITS telemetry devices were used to collect heart rate data. (A) NHP 1; (B) NHP 2; (C) NHP 3; (D) NHP 4. The grey line represents baseline (mean of the telemetry data collected in the six days prior to challenge) for each animal. The diamonds represent statistically significant heart rate values (> +3 SD [♦] above or > -3 SD [♦] below baseline) or non-significant values (♦). bpm = beats per minute.</p

Weight change for NiV-infected animals.

<p>Weights for animals on study are shown as a percent of baseline (mean of the PID-8, -1, and 0 values for that animal). Black lines and symbols represent animals that succumbed, and grey lines and symbols represent animals that survived to end of study.</p

A DNA vaccine delivered by dermal electroporation fully protects cynomolgus macaques against Lassa fever

Lassa virus (LASV) is an ambisense RNA virus in the Arenaviridae family and is the etiological agent of Lassa fever, a severe hemorrhagic disease endemic to West and Central Africa. There are no US Food and Drug Administration (FDA)-licensed vaccines available to prevent Lassa fever. in our previous studies, we developed a gene-optimized DNA vaccine that encodes the glycoprotein precursor gene of LASV (Josiah strain) and demonstrated that 3 vaccinations accompanied by dermal electroporation protected guinea pigs from LASV-associated illness and death. Here, we describe an initial efficacy experiment in cynomolgus macaque nonhuman primates (NHPs) in which we followed an identical 3-dose vaccine schedule that was successful in guinea pigs, and a follow-on experiment in which we used an accelerated vaccination strategy consisting of 2 administrations, spaced 4 weeks apart. In both studies, all of the LASV DNA-vaccinated NHPs survived challenge and none of them had measureable, sustained viremia or displayed weight loss or other disease signs post-exposure. Three of 10 mock-vaccinates survived exposure to LASV, but all of them became acutely ill post-exposure and remained chronically ill to the study end point (45 d post-exposure). Two of the 3 survivors experienced sensorineural hearing loss (described elsewhere). These results clearly demonstrate that the LASV DNA vaccine combined with dermal electroporation is a highly effective candidate for eventual use in humans