Carly M. Malavé
Jaime Lopera-Madrid
Lex G. Medina-Magües
Tonie E. Rocke
Jorge E. Osorio
20211202
In vitro expression, immunogenicity, and efficacy data from recombinant raccoon poxvirus-vectored rabies vaccine candidates tested in mice
spreadsheet
https://doi.org/10.5066/P9IERY9D
This work is part of an experimental trial to develop and assess novel recombinant raccoonpox virus (RCN) rabies vaccines in the mouse model, for potential use in bats. Briefly, our research group previously developed a recombinant RCN vaccine candidate expressing a mosaic glycoprotein (MoG) gene that protected mice and big brown bats when challenged with rabies virus (RABV). We developed two new recombinant RCN candidates expressing MoG (RCN-tPA-MoG and RCN-SS-TD-MoG) with the aim of improving RCN-MoG. We assessed and compared in vitro expression, in vivo immunogenicity, and protective efficacy in vaccinated mice challenged intracerebrally with RABV. In this data set, we share results of immunofluorescence assay quantification, recombinant virus growth curves, and antibody titer assays with mouse sera. Additionally, we share mouse weights and weight changes following RABV challenge, as well as RABV challenge study survival results. These data demonstrate that vaccination with either RCN-tPA-MoG or RCN-MoG confers adequate protection from rabies infection, and either may be a sufficient vaccine candidate for bats in future work.
These data were collected to compare potential rabies vaccine candidates by testing in cell culture and the mouse model. They can be used to as evidence for continued testing, ideally in the target species (i.e., bats).
20201001
20210801
ground condition
None planned
Madison, Wisconsin
-89.48609
-89.41189
43.07669
43.04619
ISO 19115 Topic Category
biota
None
rabies
rabies virus
RABV
raccoon poxvirus
recombinant
vaccine
A/J mice
USGS Metadata Identifier
USGS:6196c135d34eb622f691acc6
None
University of Wisconsin-Madison, Madison, WI - Hanson Biomedical Sciences
National Wildlife Health Center, Madison, WI
None. Please see 'Distribution Info' for details.
None. Users are advised to read the dataset's metadata thoroughly to understand appropriate use and data limitations.
Carly M Malave
U.S. Geological Survey
mailing address
6006 Schroeder Road
Madison
WI
53711
US
608-270-2400
608-270-2415
cmalave@usgs.gov
University of Wisconsin-Madison
Assays were repeated if standards (i.e., positive and negative controls) were not measured correctly or did not yield expected results. Spreadsheets were checked and double-checked for errors or missing information.
Yes – this data has been reviewed several times for any gross errors and inaccuracies
All data represented.
Table 1. IF Quantification
Images taken from the immunofluorescence assay were quantified using an image processing protocol described in Meza et. al (see above). Briefly, plates were photographed at 20 x magnification (70% brightness) under a fluorescent microscope (excitation wavelength 590 nm, emission wavelength 617 nm; AMG EVOSfl, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). Three equally sized fields from the center of each well were selected and photographed (left to right). Images were processed through ImageJ (version 2.1.0/1.352k) and Fiji [22,23]. First, the images were transformed into an 8-bit binary representation (every pixel stored as a single bit), indicating the presence (white) and absence (black in the background) of GFP fluorescence. Then, images were processed using the Autolocal Threshold Phansalkar plugin (size radius 1-5), and the function “Analyze Particles” was used to count the total number of fluorescent particles per field (i.e., cells expressing MoG protein). This command grouped and counted the white pixels with a general size area and circularity to capture every area of fluorescence (size area: 0–infinity; circularity: 1). A summary including the number of white particles counted in each image was generated and used in the analysis.
Meza DK, Broos A, Becker DJ, Behdenna A, Willett BJ, Viana M, Streicker DG. Predicting the presence and titer of rabies virus neutralizing antibodies from low-volume serum samples in low-containment facilities. BioRxiv. 2020 Jan 1.
20210719
Figure 3. Virus Growth Curve
Vero cells in 6-well plates were infected at an MOI of 0.5 with RCN-MoG, RCN-tPA-MoG, RCN-SS-TD-MoG, and RCN-GFP; additional wells were left uninfected to serve as a negative infection control. Cells were incubated at 37°C and harvested at 12, 24, 36 and 48 h post-inoculation. Virus titration was performed in 96-well plates using Vero cells seeded at a concentration of 104 cells/well to achieve confluency after 24 h incubation at 37°C. Plates were infected with 50 μL of a 10-fold serial dilution of recombinant virus (10-1 to 10-12) with 8 replicates per dilution and incubated for 3 days. Plates were visualized under a fluorescent microscope, where wells displaying one or more viral plaques were designated as positive. Virus titers were calculated using the Reed-Muench method (see above).
Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. American Journal of Epidemiology. 1938 May 1;27(3):493-7
20210601
Figure 4. Mouse immunogenicity
Serum samples were analyzed for detectable rabies virus neutralizing antibody (RVNA) titers using a modified micro neutralization assay (see above), based on the Rapid Fluorescent Focus Inhibition Test (see above). Briefly, mouse sera were mixed with BHK-21 cells and CVS-11 RABV in MEM-10 media in a 4-well Teflon coated slide; after incubation, slides were fixed with acetone, stained with an FITC RABV stain (Fujirebio U.S. Inc., Malvern, Pennsylvania, USA), and visualized under a fluorescent microscope. Ten microscopic fields per well were read for presence and absence of fluorescing cells, and the number of fluorescent fields per well were used to calculate the endpoint titers via the Reed-Muench method [24]. Titers were converted to international units per milliliter (IU/mL) by comparison to a standard rabies immunoglobulin (SRIG) positive control with 2 IU/mL. For the objective of this study, the positive cutoff value (greater than or equal to 0.5 IU/mL) was determined by at least 50% neutralization of the CVS-11 challenge virus (50 fluorescent foci doses) in a 1:10 dilution of the SRIG.
Smith TG, Gilbert AT. Comparison of a micro-neutralization test with the rapid fluorescent focus inhibition test for measuring rabies virus neutralizing antibodies. Tropical Medicine and Infectious Disease. 2017 Sep;2(3):24.
Smith JS, Yager PA, Baer GM. A rapid fluorescent focus inhibition test (RFFIT) for determining rabies virus-neutralizing antibody. 4th ed. Meslin FX, Kaplan MM, Koprowski H, editors. Laboratory Techniques in Rabies. Geneva: World Health Organization; 1996.
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Figure 6. Mouse survival
Mouse brains were assessed for rabies infection using the direct fluorescent antibody test (DFA). After brain impressions were fixed in acetone, slides were stained with an FITC-labelled monoclonal antibody (mAB) conjugate (Fujirebio U.S. Inc., Malvern, Pennsylvania, USA) and visualized under a fluorescent microscope, as described elsewhere (see above).
Dean DJ, Abelseth MK, Atanasiu P. The flourescent antibody test. In: Meslin M.M. Koprowski, H. FXK, editor. Laboratory Techniques in Rabies. Geneva, Switzerland: World Health Organization; 1996. pp. 83–93
20210105
Table 1. IF quantification.csv
Comma Separated Value (CSV) file containing data.
Producer Defined
Image
Name/label for the image used in the analysis
Producer Defined
image ID
RCN-MoG
RCN-MoG virus used to infect cells and produce fluorescent particles. Number of fluorescent particles counted during analysis.
Producer Defined
108
199
particles
RCN-tPA-MoG
RCN-tPA-MoG virus used to infect cells and produce fluorescent particles. Number of fluorescent particles counted during analysis.
Producer Defined
2234
2416
particles
RCN-SS-TD-MoG
RCN-SS-TD-MoG virus used to infect cells and produce fluorescent particles. Number of fluorescent particles counted during analysis.
Producer Defined
1201
1710
particles
Figure 3. Virus growth curve.csv
Comma Separated Value (CSV) file containing data.
Producer Defined
Time of virus collection (hours after infection)
Time point at which virus was collected for titration
Producer Defined
12
48
hours
RCN-MoG
RCN-MoG virus used to infect cells and produce virus plaque forming units. Virus plaque forming units counted after collection/titration.
Producer Defined
3270
4850000
Plaque forming units (pfu)
RCN-tPA-MoG
RCN-tPA-MoG virus used to infect cells and produce virus plaque forming units. Virus plaque forming units counted after collection/titration.
Producer Defined
4140
8910000
Plaque forming units (pfu)
RCN-SS-TD-MoG
RCN-SS-TD-MoG virus used to infect cells and produce virus plaque forming units. Virus plaque forming units counted after collection/titration.
Producer Defined
8220
3720000
Plaque forming units (pfu)
Figure 4. Mouse immunogenecity.csv
Comma Separated Value (CSV) file containing data.
Producer Defined
Mouse ID
Number representing each individual animal
Producer Defined
Numeric ID
Treatment
Describes the virus/control substance administered to animals to produce antibodies
Producer Defined
RCN-MoG
RCN-MoG
Producer defined
RCN-tPA-MoG
RCN-tPA-MoG
Producer defined
RCN-SS-TD-MoG
RCN-SS-TD-MoG
Producer defined
PBS
phosphate-buffered saline
Producer defined
14 dpv
Rabies virus neutralizing antibody (RVNA) titer of individual at time of collection at 14 days post vaccination
Producer Defined
0.02
2.4
IU/mL (International units per milliliter)
27 dpv
Rabies virus neutralizing antibody (RVNA) titer of individual at time of collection at 27 days post vaccination
Producer Defined
0.02
4.47
IU/mL (International units per milliliter)
Terminal
Rabies virus neutralizing antibody (RVNA) titer of individual at time of collection at end of animals's life – not necessarily the same date for each animal.
Producer Defined
0.02
147.1
IU/mL (International units per milliliter)
Figure 6. Mouse survival.csv
Comma Separated Value (CSV) file containing data.
Producer Defined
Mouse ID
Number representing each individual animal
Producer Defined
numeric ID
Day of death (euthansia or end of study)
Day after virus challenge when animal was euthanized or died
Producer Defined
N/A
No Data
Producer defined
7
14
days
RCN-MoG
Received RCN-MoG treatment. Numerical value indicating that an animal survived the virus challenge (0) or did not (1).
Producer Defined
<< empty cell >>
Mouse not in treatment group
Producer defined
0
survived
Producer defined
1
did not survive
Producer defined
RCN-tPA-MoG
Received RCN-tPA-MoG treatment. Numerical value indicating that an animal survived the virus challenge (0) or did not (1).
Producer Defined
<< empty cell >>
Mouse not in treatment group
Producer defined
0
survived
Producer defined
RCN-SS-TD-MoG
Received RCN-SS-TD-MoG treatment. Numerical value indicating that an animal survived the virus challenge (0) or did not (1).
Producer Defined
<< empty cell >>
Mouse not in treatment group
Producer defined
0
survived
Producer defined
1
did not survive
Producer defined
N/A
N/A
Producer defined
PBS
Received phosphate-buffered saline treatment. Numerical value indicating that an animal survived the virus challenge (0) or did not (1).
Producer Defined
<< empty cell >>
No Data
Producer defined
1
did not survive
Producer defined
0
survived
Producer defined
Figure 5A. Mouse weight change.csv
Comma Separated Value (CSV) file containing data.
Producer Defined
Mouse ID
Number representing each individual animal
Producer Defined
numeric ID
Treatment
Describes the virus/control substance administered to animals to prior to virus challenge
Producer Defined
RCN-MoG
RCN-MoG treatment
Producer defined
RCN-tPA-MoG
RCN-tPA-MoG treatment
Producer defined
RCN-SS-TD-MoG
RCN-SS-TD-MoG treatment
Producer defined
PBS
phosphate-buffered saline
Producer defined
0
Weight of mouse 0 days post challenge
Producer Defined
14.1
19.5
Grams
1
Weight of mouse 1 day post challenge
Producer Defined
14.1
19.5
Grams
2
Weight of mouse 2 days post challenge
Producer Defined
14.2
19.9
Grams
3
Weight of mouse 3 days post challenge
Producer Defined
14.1
19.7
Grams
4
Weight of mouse 4 days post challenge
Producer Defined
14.1
19.7
Grams
5
Weight of mouse 5 days post challenge
Producer Defined
13.9
19.6
Grams
6
Weight of mouse 6 days post challenge
Producer Defined
14.0
19.9
Grams
7
Weight of mouse 7 days post challenge
Producer Defined
12.7
20.3
Grams
8
Weight of mouse 8 days post challenge
Producer Defined
<< empty cell >>
No Data
Producer defined
12.0
19.8
Grams
9
Weight of mouse 9 days post challenge
Producer Defined
<< empty cell >>
No Data
Producer defined
10.8
19.6
Grams
10
Weight of mouse 10 days post challenge
Producer Defined
<< empty cell >>
No Data
Producer defined
14.7
19.5
Grams
11
Weight of mouse 11 days post challenge
Producer Defined
<< empty cell >>
No Data
Producer defined
15.4
19.5
Grams
12
Weight of mouse 12 days post challenge
Producer Defined
<< empty cell >>
No Data
Producer defined
15.7
20.0
Grams
13
Weight of mouse 13 days post challenge
Producer Defined
<< empty cell >>
No Data
Producer defined
15.5
19.7
Grams
14
Weight of mouse 14 days post challenge
Producer Defined
<< empty cell >>
No Data
Producer defined
15.4
20.3
Grams
Figure 5B. Mouse weight change.csv
Comma Separated Value (CSV) file containing data.
Producer Defined
Mouse ID
Number representing each individual animal
Producer Defined
numeric ID
Treatment
Describes the virus/control substance administered to animals to prior to virus challenge
Producer Defined
RCN-MoG
RCN-MoG treatment
Producer defined
RCN-tPA-MoG
RCN-tPA-MoG treatment
Producer defined
RCN-SS-TD-MoG
RCN-SS-TD-MoG treatment
Producer defined
PBS
phosphate-buffered saline
Producer defined
0
Percentage of weight gained or lost on day 0 when compared to weight on day 0
Producer Defined
0
0
Percentage (%)
1
Percentage of weight gained or lost on day 1 when compared to weight on day 0
Producer Defined
-8.426966292000001
4.972375691
Percentage (%)
2
Percentage of weight gained or lost on day 2 when compared to weight on day 0
Producer Defined
-10.674157300000001
6.32183908
Percentage (%)
3
Percentage of weight gained or lost on day 3 when compared to weight on day 0
Producer Defined
-12.92134831
5.617977528
Percentage (%)
4
Percentage of weight gained or lost on day 4 when compared to weight on day 0
Producer Defined
-8.988764045
5.1428571430000005
Percentage (%)
5
Percentage of weight gained or lost on day 5 when compared to weight on day 0
Producer Defined
-14.60674157
6.077348066
Percentage (%)
6
Percentage of weight gained or lost on day 6 when compared to weight on day 0
Producer Defined
-19.66292135
7.182320442000001
Percentage (%)
7
Percentage of weight gained or lost on day 7 when compared to weight on day 0
Producer Defined
-26.96629213
8.839779006
Percentage (%)
8
Percentage of weight gained or lost on day 8 when compared to weight on day 0
Producer Defined
<< empty cell >>
No Data
Producer defined
-22.64150943
7.428571429
Percentage (%)
9
Percentage of weight gained or lost on day 9 when compared to weight on day 0
Producer Defined
<< empty cell >>
No Data
Producer defined
-23.91304348
7.428571429
Percentage (%)
10
Percentage of weight gained or lost on day 10 when compared to weight on day 0
Producer Defined
<< empty cell >>
No Data
Producer defined
-19.23076923
6.179775281
Percentage (%)
11
Percentage of weight gained or lost on day 11 when compared to weight on day 0
Producer Defined
<< empty cell >>
No Data
Producer defined
-19.37172775
5.617977528
Percentage (%)
12
Percentage of weight gained or lost on day 12 when compared to weight on day 0
Producer Defined
<< empty cell >>
No Data
Producer defined
-17.27748691
7.865168539
Percentage (%)
13
Percentage of weight gained or lost on day 13 when compared to weight on day 0
Producer Defined
<< empty cell >>
No Data
Producer defined
-18.84816754
6.179775281
Percentage (%)
14
Percentage of weight gained or lost on day 14 when compared to weight on day 0
Producer Defined
<< empty cell >>
No Data
Producer defined
-17.27748691
6.741573034
Percentage (%)
U.S. Geological Survey
GS ScienceBase
mailing address
Denver Federal Center, Building 810, Mail Stop 302
Denver
CO
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United States
1-888-275-8747
sciencebase@usgs.gov
Unless otherwise stated, all data, metadata and related materials are considered to satisfy the quality standards relative to the purpose for which the data were collected. Although these data and associated metadata have been reviewed for accuracy and completeness and approved for release by the U.S. Geological Survey (USGS), no warranty expressed or implied is made regarding the display or utility of the data on any other system or for general or scientific purposes, nor shall the act of distribution constitute any such warranty.
Digital Data
https://doi.org/10.5066/P9IERY9D
None
20211202
Carly M Malave
U.S. Geological Survey
mailing address
6006 Schroeder Road
Madison
WI
53711
US
608-270-2400
608-270-2415
cmalave@usgs.gov
FGDC Biological Data Profile of the Content Standard for Digital Geospatial Metadata
FGDC-STD-001.1-1999