Needle-free delivery of measles virus vaccine to the lower respiratory tract of non-human primates elicits optimal immunity and protection

Publication history: Accepted - 8 June 2017; Published online - 1 August 2017.Needle-free measles virus vaccination by aerosol inhalation has many potential benefits. The current standard route of vaccination is subcutaneous injection, whereas measles virus is an airborne pathogen. However, the target cells that support replication of liveattenuated measles virus vaccines in the respiratory tract are largely unknown. The aims of this study were to assess the in vivo tropism of live-attenuated measles virus and determine whether respiratory measles virus vaccination should target the upper or lower respiratory tract. Four groups of twelve cynomolgus macaques were immunized with 104 TCID50 of recombinant measles virus vaccine strain Edmonston-Zagreb expressing enhanced green fluorescent protein. The vaccine virus was grown in MRC-5 cells and formulated with identical stabilizers and excipients as used in the commercial MVEZ vaccine produced by the Serum Institute of India. Animals were immunized by hypodermic injection, intra-tracheal inoculation, intra-nasal instillation, or aerosol inhalation. In each group six animals were euthanized at early time points post-vaccination, whereas the other six were followed for 14 months to assess immunogenicity and protection from challenge infection with wild-type measles virus. At early time-points, enhanced green fluorescent protein-positive measles virus-infected cells were detected locally in the muscle, nasal tissues, lungs, and draining lymph nodes. Systemic vaccine virus replication and viremia were virtually absent. Infected macrophages, dendritic cells and tissueresident lymphocytes predominated. Exclusive delivery of vaccine virus to the lower respiratory tract resulted in highest immunogenicity and protection. This study sheds light on the tropism of a live-attenuated measles virus vaccine and identifies the alveolar spaces as the optimal site for respiratory delivery of measles virus vaccine.This study was funded by the Foundation for the National Institutes of Health, through the Bill and Melinda Gates Foundation Grand Challenges in Global Health initiative (grant number: DUPREX09GCGH0)

Use of influenza A viruses expressing reporter genes to assess the frequency of double infections in vitro

Exchange of gene segments between mammalian and avian influenza A viruses may lead to the emergence of potential pandemic influenza viruses. Since co-infection of single cells with two viruses is a prerequisite for reassortment to take place, we assessed frequencies of doubleinfection in vitro using influenza A/H5N1 and A/H1N1 viruses expressing the reporter genes eGFP or mCherry. Double-infected A549 and Madin-Darby canine kidney cells were detected by confocal microscopy and flow cytometry

Complete genome sequence of phocine distemper virus isolated from a harbor seal (Phoca vitulina) during the 1988 North Sea epidemic

Phocine distemper virus (PDV) was identified as the cause of a large morbillivirus outbreak among harbor seals in the North Sea in 1988. PDV is a member of the family Paramyxoviridae, genus Morbillivirus. Until now, no full-genome sequence of PDV has been available

Plasmodium CDP-DAG synthase: An atypical gene with an essential N-terminal extension

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Statistical Model To Evaluate In Vivo Activities of Antimalarial Drugs in a Plasmodium cynomolgi-Macaque Model for Plasmodium vivax Malaria▿ ‡

Preclinical animal models informing antimalarial drug development are scarce. We have used asexual erythrocytic Plasmodium cynomolgi infections of rhesus macaques to model Plasmodium vivax during preclinical development of compounds targeting parasite phospholipid synthesis. Using this malaria model, we accumulated data confirming highly reproducible infection patterns, with self-curing parasite peaks reproducibly preceding recrudescence peaks. We applied nonlinear mixed-effect (NLME) models, estimating treatment effects in three drug studies: G25 (injected) and the bisthiazolium prodrugs TE4gt and TE3 (oral). All compounds fully cured P. cynomolgi-infected macaques, with significant effects on parasitemia height and time of peak. Although all three TE3 doses tested were fully curative, NLME models discriminated dose-dependent differential pharmacological antimalarial activity. By applying NLME modeling treatment effects are readily quantified. Such drug development studies are more informative and contribute to reduction and refinement in animal experimentation

Measles immune suppression:lessons from the Macaque Model

Measles remains a significant childhood disease, and is associated with a transient immune suppression. Paradoxically, measles virus (MV) infection also induces robust MV-specific immune responses. Current hypotheses for the mechanism underlying measles immune suppression focus on functional impairment of lymphocytes or antigen-presenting cells, caused by infection with or exposure to MV. We have generated stable recombinant MVs that express enhanced green fluorescent protein, and remain virulent in non-human primates. By performing a comprehensive study of virological, immunological, hematological and histopathological observations made in animals euthanized at different time points after MV infection, we developed a model explaining measles immune suppression which fits with the "measles paradox". Here we show that MV preferentially infects CD45RA-memory T-lymphocytes and follicular B-lymphocytes, resulting in high infection levels in these populations. After the peak of viremia MV-infected lymphocytes were cleared within days, followed by immune activation and lymph node enlargement. During this period tuberculin-specific T-lymphocyte responses disappeared, whilst strong MV-specific T-lymphocyte responses emerged. Histopathological analysis of lymphoid tissues showed lymphocyte depletion in the B- and T-cell areas in the absence of apoptotic cells, paralleled by infiltration of T-lymphocytes into B-cell follicles and reappearance of proliferating cells. Our findings indicate an immune-mediated clearance of MV-infected CD45RA-memory T-lymphocytes and follicular B-lymphocytes, which causes temporary immunological amnesia. The rapid oligoclonal expansion of MV-specific lymphocytes and bystander cells masks this depletion, explaining the short duration of measles lymphopenia yet long duration of immune suppression

Histology and immunohistochemistry of lymphoid tissues obtained from macaques euthanized between 5 and 15 d.p.i.

<p>Serial sections were stained for histological changes (H&E), MV infection (EGFP), B-lymphocytes (CD20), T-lymphocytes (CD3) or proliferating cells (Ki67). Multiple lymphoid tissues from multiple animals collected at each time-point were analyzed, panels shown are representative for the tissues that have been examined. The color intensity of the blue bars below the photomicrographs indicates the relative levels of viremia, lymphocyte depletion or proliferation. The green and red bars at the bottom indicate the appearance and disappearance of viremia and rash and correspond to the bars in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002885#ppat-1002885-g005" target="_blank">Figure 5B</a>.</p

Susceptibility of human or macaque T-lymphocyte subsets to <i>in vitro</i> MV infection.

<p>(A) Human or macaque PBMC were sorted into naive (CD45RA⁺, Tⁿ) or memory (CD45RA⁻, T^M) CD4⁺ or CD8⁺ T-lymphocytes, and infected with MV <i>in vitro</i>. Percentages MV-infected T-lymphocytes were determined 2 d.p.i. by measuring EGFP fluorescence by flow cytometry. CD4⁺ (human and macaque) and CD8⁺ (human only) T^M were significantly more susceptible to MV infection than the corresponding Tⁿ. For macaque CD8⁺ T-lymphocytes the difference was significant in two out of three experiments; (B) Unsorted human and macaque PBMC were infected, MV infection percentages in the different T-lymphocyte subsets were determined 2 d.p.i. by flow cytometry. Both in human and macaque PBMC the CD4⁺ and CD8⁺ T^CM and T^EM were significantly more susceptible to MV infection than the corresponding Tⁿ subpopulations. In addition, CD4⁺ T^EM and, to a lesser extent, CD8⁺ T^EM proved more susceptible to MV infection than T^CM. (C–F) Levels of CD150 expression on the different T-lymphocyte subsets in human and macaque PBMC. (C) PBMC collected from human or macaque donors were stained for memory markers as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002885#ppat.1002885.s002" target="_blank">Figure S1</a>, in combination with an IgG1 isotype control or CD150^FITC staining. CD150 expression on the different subsets is shown as geometric mean fluorescence intensity (Gmean FI) ± SD. Both for humans and macaques CD150 expression on CD4⁺ and CD8⁺ T^CM and T^EM was significantly higher than on Tⁿ. Interestingly, in CD4⁺ T-lymphocytes CD150 expression was significantly higher on T^EM than on T^CM, whereas in CD8⁺ T-lymphocytes an inverse pattern was observed. (D–F) An IgG1 isotype control was used to determine the level of background staining, and is shown in combination with the CD150 staining for each subset. **, <i>P</i><0.01. *, <i>P</i><0.05. Experiments were performed with sorted cells from 3 macaque and 2 human donors, and unsorted cells from 4 macaque and 5 human donors. Data are shown as means ± standard deviation (SD) of representative donors.</p

MV infection causes temporary immunological amnesia.

<p>(A) T-lymphocyte responses to PPD and MV were measured by IFN-γ production after <i>in vitro</i> stimulation of PBMC collected from 4 BCG-vaccinated macaques. Measurements were performed in triplicate, graphs shows means ± SD; (B) Mantoux tests were performed 7 days before (n = 4) and 8 (n = 2) or 10 (n = 2) days after MV infection. Images were collected 3 days after intra-dermal injection with tuberculin. Before MV infection classical delayed-type hypersensitivity responses were observed, associated with diffuse swelling and redness (indicated by arrows), after MV only a small localized papule was observed (indicated by arrow). H&E and CD3 staining of the corresponding skin tissues showed infiltration of T-lymphocytes in the dermis of the pre-infection Mantoux response, which was absent after MV infection. Representative images from 4 animals are shown.</p

MV infects high percentages of B-lymphocytes and CD45RA⁻ memory T-lymphocytes.

<p>(A–D) Macroscopic detection of EGFP in lymphoid tissues of the gastro-intestinal tract in three different macaques: mesenteric lymph nodes (A), gut-associated lymphoid tissue (GALT) (B and C), including the Peyer's patches (C and D). Panel D is an enlargement of panel C (indicated by asterisk); (E–H) MV infection percentages in lymphocyte subsets during the approximate peak viremia. T-lymphocyte subpopulations were identified as Tⁿ (CD45RA⁺), T^CM (CD45RA⁻CCR7⁺) or T^EM (CD45RA⁻CCR7⁻), B-lymphocytes were identified as Bⁿ (CD27⁻IgD⁺) or B^M (CD27⁺IgD⁻). Box plots were chosen since the data were not normally distributed, and show the median infection percentages with the 25^th–75^th percentiles, error bars indicate the 10^th–90^th percentiles, dots the 5^th–95^th percentiles. The 10^th–90^th percentiles and 5^th–95^th percentiles are only shown if the number of observations is at least ten; **, <i>P</i><0.01. *, <i>P</i><0.05. In panel E, F and G 14 animals were included; in panel H 3 animals were included.</p