Mucosal Immunization of Cynomolgus Macaques with the VSVΔG/ZEBOVGP Vaccine Stimulates Strong Ebola GP-Specific Immune Responses

(ZEBOV) produces a lethal viral hemorrhagic fever in humans and non-human primates.We demonstrate that the VSVΔG/ZEBOVGP vaccine given 28 days pre-challenge either intranasally (IN), orally (OR), or intramuscularly (IM) protects non-human primates against a lethal systemic challenge of ZEBOV, and induces cellular and humoral immune responses. We demonstrated that ZEBOVGP-specific T-cell and humoral responses induced in the IN and OR groups, following an immunization and challenge, produced the most IFN-γ and IL-2 secreting cells, and long term memory responses.We have shown conclusively that mucosal immunization can protect from systemic ZEBOV challenge and that mucosal delivery, particularly IN immunization, seems to be more potent than IM injection in the immune parameters we have tested. Mucosal immunization would be a huge benefit in any emergency mass vaccination campaign during a natural outbreak, or following intentional release, or for mucosal immunization of great apes in the wild

Ebola Virion Attachment and Entry into Human Macrophages Profoundly Effects Early Cellular Gene Expression

Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP1,2) is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP1,2 (VLPVP40-GP) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLPVP40 (particles lacking GP1,2) caused an aberrant response. This suggests that GP1,2 binding to macrophages plays an important role in the immediate cellular response

Lethal Influenza Virus Infection in Macaques Is Associated with Early Dysregulation of Inflammatory Related Genes

The enormous toll on human life during the 1918–1919 Spanish influenza pandemic is a constant reminder of the potential lethality of influenza viruses. With the declaration by the World Health Organization of a new H1N1 influenza virus pandemic, and with continued human cases of highly pathogenic H5N1 avian influenza virus infection, a better understanding of the host response to highly pathogenic influenza viruses is essential. To this end, we compared pathology and global gene expression profiles in bronchial tissue from macaques infected with either the reconstructed 1918 pandemic virus or the highly pathogenic avian H5N1 virus A/Vietnam/1203/04. Severe pathology was observed in respiratory tissues from 1918 virus-infected animals as early as 12 hours after infection, and pathology steadily increased at later time points. Although tissues from animals infected with A/Vietnam/1203/04 also showed clear signs of pathology early on, less pathology was observed at later time points, and there was evidence of tissue repair. Global transcriptional profiles revealed that specific groups of genes associated with inflammation and cell death were up-regulated in bronchial tissues from animals infected with the 1918 virus but down-regulated in animals infected with A/Vietnam/1203/04. Importantly, the 1918 virus up-regulated key components of the inflammasome, NLRP3 and IL-1β, whereas these genes were down-regulated by A/Vietnam/1203/04 early after infection. TUNEL assays revealed that both viruses elicited an apoptotic response in lungs and bronchi, although the response occurred earlier during 1918 virus infection. Our findings suggest that the severity of disease in 1918 virus-infected macaques is a consequence of the early up-regulation of cell death and inflammatory related genes, in which additive or synergistic effects likely dictate the severity of tissue damage

Absolute white blood cell numbers do not decrease in immunized animals after Zaire ebolavirus challenge.

<p>Whole blood from cynomolgus macaques immunized either orally (OR; n = 4), intranasally (IN; n = 4) or intramuscularly (IM; n = 2) was stained for lympocytes (CD3⁺, CD4⁺, CD8⁺), monocytes (CD14⁺). Control animals (n = 2) received non protective VSVΔG/MARVGP. The absolute numbers in blood was determined for the each of the monkeys by flow cytometry on days 0, 3 and 6 post-challenge with ZEBOV. The average for each inoculation route is represented.</p

Strong humoral IgM, IgA, IgG responses are induced in the IM, OR, and IN immunization routes.

<p>A ZEBOV VLP ELISA was utilized to determine the ZEBOV-specific IgA (a), IgG (b), IgM (c) titres in post-vaccination and post-challenge sera from cynomolgus macaques immunized either orally (OR; n = 4), intranasally (IN; n = 4) or intramuscularly (IM; n = 2). Titres are presented as endpoint dilutions of the average value per group. Table (d) illustrates fold increases in peak titres post-challenge compared to post-vaccination for IgA and IgG. IgM is not shown as there were no increases post-challenge.</p

Blood Hematology Results for Cynomolgus Monkeys after vaccination and challenge with Ebola Virus.

<p>Pre-vaccination test results for each animal (n = 12) were used to establish an average for each parameter. Post-vaccination and -challenge values were compared to the pre-vaccination average. Significant changes in comparison to this average are indicated (*). When values for each animal were compared to its' own pre-vaccination values, no changes (NC) were observed in any of the parameters.</p><p>HCT = hematocrit, RBC = red blood cells, HGB = hemaglobin, MCV = mean corpuscular volume, % RDW = red cell distribution width, MCHC = mean corpuscular hemoglobin concentration, MCH = mean corpuscular hemoglobin, MPV = mean platelet volume, PCT = platelet-crit, % PDW = platelet distribution width. Post Vac = post vaccination, Post Chall = post challenge, OR = oral, IN = intranasal, IM = intramuscular.</p

Survival of vaccinated cynomolgus macaques.

<p>A) <i>Flow chart of experimental design.</i> Arrows indicate vaccination and challenge date. Tick marks indicate sampling days. B) <i>Kaplan-Meier survival curve for cynomolgus macaques immunized by different routes and challenged with Zaire ebolavirus</i>. Cynomolgus macaques were immunized orally (OR; n = 4), intranasally (IN; n = 4) or intramuscularly (IM; n = 2) with 2×10⁷ PFU of VSVΔG/ZEBOVGP, or injected intramuscularly with VSVΔG/MARVGP (control; n = 2). All animals were challenged 28 days later with 1000 PFU ZEBOV. The animals were scored daily for fever, macular rashes, lethargy, and unresponsiveness.</p

PBMCs from immunized animals produce IFN-γ or IL-2 in response to ZEBOVGP peptides.

<p>ZEBOVGP-specific IFN-γ or IL-2 secreting cells from PBMCs were detected by either an (A) IFN-γ or (B) IL-2 ELISPOT assay. PBMCs were obtained from cynomolgus macaques immunized with VSVΔG/ZEBOVGP either orally (OR; n = 4), intranasally (IN; n = 4), or intramuscularly (IM; n = 2). The control NHPs (n = 2) were immunized IM with the heterologous VSVΔG/MARVGP. PBMCs were incubated with 1.5 µg/ml of peptides spanning the entire ZEBOV glycoprotein. Bars represent the average number of ZEBOVGP-specific IFN-γ or IL-2 secreting cells detected in each immunized group.</p

Neutralizing antibodies generated by the various immunization routes.

<p>ZEBOV GP-specific neutralizing antibodies in the sera from NHPs vaccinated either (A) intramuscularly (IM), (B) orally (OR), or (C) intranasally (IN), were investigated for their ability to inhibit infection by ZEBOV-GFP. NHP sera from days 0 and 21 post-vaccination were incubated for 1 hour with ZEBOV-GFP before being added to a monolayer of Vero cells. The positive control was ZEBOV-GFP in DMEM without sera. The level of GFP fluorescence of ZEBOV-GFP infected cells was determined by flow cytometry. The percent reduction of infection by ZEBOV-GFP was calculated as follows: % ZEBOV-GFP reduction = (1−(Test samples/Positive control))×100). The day 21 results for each NHP are displayed and have been normalized by subtracting each NHP's day 0 results from the day 21 results. The numbers in the legend represent the dilution of the NHP sera that was added to ZEBOV-GFP.</p