Immunogenicity of a Candidate Live Attenuated Vaccine for Rift Valley Fever Virus with a Two-Segmented Genome

Abstract

Rift Valley fever virus (RVFV) is an emerging arbovirus that affects both ruminants and humans. RVFV causes severe and recurrent outbreaks in Africa and the Arabian Peninsula with a significant risk for emergence into new locations. Although there are a variety of RVFV veterinary vaccines for use in endemic areas, there is currently no licensed vaccine for human use; therefore, there is a need to develop and assess new vaccines. Herein, we report a live-attenuated recombinant vaccine candidate for RVFV, based on the previously described genomic reconfiguration of the conditionally licensed MP12 vaccine. There are two general strategies used to develop live-attenuated RVFV vaccines, one being serial passage of wild-type RVFV strains to select attenuated mutants such as Smithburn, Clone 13, and MP12 vaccine strains. The second strategy has utilized reverse genetics to attenuate RVFV strains by introducing deletions or insertions within the viral genome. The novel candidate vaccine characterized in this report contains a two-segmented genome that lacks the medium viral segment (M) and two virulence genes (nonstructural small and nonstructural medium). The vaccine candidate, named r2segMP12, was evaluated for the production of neutralizing antibodies to RVFV in outbred CD-1 mice. The immune response induced by the r2segMP12 vaccine candidate was directly compared to the immune response induced by the rMP12 parental strain vaccine. Our study demonstrated that a single immunization with the r2segMP12 vaccine candidate at 105 plaque-forming units elicited a higher neutralizing antibody response than the rMP12 vaccine at the same vaccination titer without the need for a booster.

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Immunogenicity of a Candidate Live Attenuated
Vaccine for Rift Valley Fever Virus
with a Two-Segmented Genome
Victoria B. Ayers,
1,2
Yan-Jang S. Huang,
1,2
James I. Dunlop,
3
Alain Kohl,
3
Benjamin Brennan,
3
Stephen Higgs,
1,2
and Dana L. Vanlandingham
1,2
Abstract
Rift Valley fever virus (RVFV) is an emerging arbovirus that affects both ruminants and humans. RVFV causes
severe and recurrent outbreaks in Africa and the Arabian Peninsula with a significant risk for emergence into
new locations. Although there are a variety of RVFV veterinary vaccines for use in endemic areas, there is
currently no licensed vaccine for human use; therefore, there is a need to develop and assess new vaccines.
Herein, we report a live-attenuated recombinant vaccine candidate for RVFV, based on the previously described
genomic reconfiguration of the conditionally licensed MP12 vaccine. There are two general strategies used to
develop live-attenuated RVFV vaccines, one being serial passage of wild-type RVFV strains to select atten-
uated mutants such as Smithburn, Clone 13, and MP12 vaccine strains. The second strategy has utilized reverse
genetics to attenuate RVFV strains by introducing deletions or insertions within the viral genome. The novel
candidate vaccine characterized in this report contains a two-segmented genome that lacks the medium viral
segment (M) and two virulence genes (nonstructural small and nonstructural medium). The vaccine candidate,
named r2segMP12, was evaluated for the production of neutralizing antibodies to RVFV in outbred CD-1 mice.
The immune response induced by the r2segMP12 vaccine candidate was directly compared to the immune
response induced by the rMP12 parental strain vaccine. Our study demonstrated that a single immunization with
the r2segMP12 vaccine candidate at 10
5
plaque-forming units elicited a higher neutralizing antibody response
than the rMP12 vaccine at the same vaccination titer without the need for a booster.
Keywords: Rift Valley fever virus, RVFV, MP12, live attenuated vaccine, double deletion
Introduction
Rift Valley fever virus (RVFV; Phenuiviridae,
Phlebovirus) is a clinically important mosquito-borne
pathogen causing disease in both humans and ruminants.
Although most humans have no clinical sign, others develop
flu-like symptoms with headaches, fever, or myalgia (Hart-
man, 2017; Laughlin et al, 1979; Wichgers Schreur et al,
2020), and around 1% of infections can progress to life-
threatening diseases, including encephalitis, hemorrhagic
fever, or thrombosis (Ikegami and Makino, 2011). While
humans are considered dead-end hosts for RVFV (Chevalier
et al, 2010), ruminants, especially sheep and goats, act as
amplifying hosts (Hartman, 2017). In livestock, death from
the disease is most commonly caused by abortion storms with
abortion rates of up to 100% (Hartman, 2017; Wichgers
1
Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, USA.
2
Biosecurity Research Institute, Kansas State University, Manhattan, Kansas, USA.
3
MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
ªVictoria B. Ayers et al. 2022; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the
Creative Commons License [CC-BY] (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
VIRAL IMMUNOLOGY
Volume 00, Number 00, 2022
Mary Ann Liebert, Inc.
Pp. 1–8
DOI: 10.1089/vim.2022.0104
1

Schreur et al, 2021). Other clinical signs in livestock
for RVFV include hyperthermia, nasal and ocular secre-
tions, and/or abdominal colic (Ikegami, 2017; Kwasnik et al,
2021).
The epidemiology of RVFV is multifactorial involving
complex relationships and dynamics between ruminants,
humans, and mosquitoes (Hartman, 2017). Transmission to
livestock and humans usually occurs through the bite of an
infected mosquito or by direct contact with infected tissues,
blood, or bodily fluids of infected animals. Infections may
also result by exposure to the virus through aerosolization
(Kwasnik et al, 2021; Pepin et al, 2010). Although the virus
is endemic in sub-Saharan Africa and the Arabian Peninsula,
susceptible ruminants and mosquito vectors are found in
many nonendemic countries.
Sheep, goats, and cattle are the main ruminants that pose
a risk of causing RVFV outbreaks, especially if involved in
importation from endemic countries (Abdo-Salem et al,
2011; Chevalier et al, 2010; Shoemaker et al, 2002). In
addition to livestock, mosquito vectors, mainly of the Aedes
and Culex genera, increase the likelihood of RVFV dispersal
and establishment in nonendemic regions ( Javelle et al,
2020).
Vaccination is the most effective method for preventing
and controlling RVFV outbreaks (Ikegami and Makino,
2009). Currently, there is no licensed vaccine or antiviral
treatment for humans or animals in nonendemic countries
(Faburay et al, 2017). Multiple veterinary vaccines are
available and commonly used in livestock in endemic
counties, including the Smithburn strain and the Clone 13
strain (Alhaj, 2016). The Smithburn strain was developed in
1971 by serial intracerebral passage in mice (Alhaj, 2016;
Smithburn, 1949). Although the Smithburn vaccine is im-
munogenic, it exhibits a partially attenuated phenotype and
cannot be used for the immunization of young and pregnant
ruminants (Botros et al, 2006; Coetzer and Barnard, 1977;
Ikegami and Makino, 2009; Kamal, 2009).
The Clone 13 strain is a naturally attenuated RVFV strain
that contains a deletion in the nonstructural small (NSs)
gene (Muller et al, 1995). The Clone 13 strain vaccine has
not only been shown to be safe and effective in lambs,
cattle, and pregnant ewes (Dungu et al., 2010; Makoschey
et al, 2016) but is also partially attenuated, as observed with
vertical transmission and teratogenic effects in ewes after
the administration of high doses (Makoschey et al, 2016).
The MP12 vaccine is conditionally licensed for use in ru-
minants in the United States (Ikegami et al, 2015; Miller
et al, 2015), and was produced through serial passage of
the ZH548 RVFV strain in the presence of 5-flurouracil
(Caplen et al, 1985). The virus was found to be attenuated
and protective in mice, lambs, and calves (Caplen et al,
1985; Wilson et al, 2014).
Several approaches have been taken to address the limi-
tations of the available candidate RVFV vaccines (Billecocq
et al, 2008; Dunlop et al, 2018; Habjan et al, 2008; Ikegami
et al, 2006) through the gene deletions of one or both vir-
ulence factors (NSs and non-structural medium [NSm]).
NSs is a nonstructural protein that facilitates evasion of the
host innate immune system (Brennan et al, 2014), while the
NSm nonstructural protein promotes suppression of apo-
ptosis in infected hosts (Ikegami and Makino, 2011). More
importantly, the deletion of NSm has previously resulted in
the reduced ability of RVFV to infect, replicate, and dis-
seminate from the midgut epithelial cells in Aedes mos-
quitoes (Kading et al, 2014).
Brennan et al developed a candidate vaccine based on the
attenuated MP12 strain that lacks the NSs and NSm genes in
a reconfigured two-segmented genome, designated r2segMP12
(Brennan et al, 2011). The rationally designed recombinant
r2segMP12 strain aims to further enhance the safety profile
of the MP12 strain based on previously published work
(Bird et al, 2008; Ikegami et al, 2006; Won et al, 2007; Won
et al, 2006).
In this study, the immunogenicity of the r2segMP12
vaccine candidate was evaluated by quantifying the serum
neutralizing activity in CD-1 mice. Groups of mice were
subcutaneously inoculated with different titers of the vac-
cine candidate on day 0, followed by a booster dose on day
21 after initial immunization. Serum samples were collected
at 20 and 42 days after initial immunization and evaluated
using plaque reduction neutralization tests (PRNT) for a
neutralizing antibody titer at or above the threshold antibody
level for protection.
The neutralizing antibody response produced by the
different titers of r2segMP12 was compared between the
use of a single dose versus a single dose followed by a
booster dose. Neutralizing antibodies produced following
the r2segMP12 vaccine and the rMP12 parental strain vac-
cine were also examined. Taken together, these data dem-
onstrated that the double deletion of NSs and NSm genes
does not reduce the immunogenicity of the MP12 vaccine
strain.
Materials and Methods
Cell lines and viruses
The r2segMP12 recombinant vaccine candidate for RVFV
was produced in a previously published study as summarized
in Figure 1 (Brennan et al, 2011). The vaccine candidate was
propagated and titered in African green monkey kidney epi-
thelial (Vero76) cells maintained at 37C in Leibovitz’s L-15
media (Thermo Fisher Scientific, Waltham, MA) supple-
mented with 10% fetal bovine serum (Thermo Fisher Scien-
tific), 10% tryptose phosphate broth (Sigma-Aldrich, St. Louis,
MO), penicillin/streptomycin (Thermo Fisher Scientific), and
L-glutamine (Thermo Fisher Scientific) as previously de-
scribed (Ayers et al, 2018). The Vero76 cell line was obtained
from the collection of Instituto Conmemorativo Gorgas de
Estudios de la Salud. The rMP12 parental strain vaccine was
generated using a control rescue experiment as previously
described (Brennan et al, 2011); and was then propagated and
titered in Vero76 cells for use as a positive control (Caplen
et al, 1985). Viral titers were determined using plaque assay as
previously described (Nuckols et al, 2015).
Animals and immunizations
The following experimental procedures and handling of
live animals were approved by the Kansas State University
Institutional Animal Care and Use Committee. All methods
were carried out in accordance with the approved proto-
col and relevant regulations. To determine the immunization
regimens required to elicit protective neutralizing anti-
body responses for the r2segMP12 vaccine candidate, fifty
2 AYERS ET AL.

3–4-week-old, outbred CD-1 mice (Charles River, Raleigh,
NC) were subcutaneously immunized with one of the fol-
lowing: r2segMP12 vaccine candidate, rMP12 parental strain
vaccine, or sterile L-15 media. Animals were randomly
assigned into 10 groups of 5, representing 8 experimental
regimens, using increasing doses (10
3
,10
4
,or10
5
plaque-
forming unit [PFU]) of infectious viruses in a single im-
munization or two immunizations (Table 1).
The regimen of a single immunization administered at an
increasing dosage per group (n=5) was included to deter-
mine the correlation of neutralizing antibodies produced
by different dosages of the r2segMP12 strain. Three groups
of mice (n=5) received a second immunization of the
r2segMP12 strain to evaluate the effect of a booster im-
munization using the dose of the vaccine that produced the
highest neutralizing antibody titer after the primary vaccine
(10
5
PFU). In addition to the experimental groups that re-
ceived the r2segMP12 vaccine candidate, four additional
groups (n=5) were designated as control groups, with two
positive control groups receiving the rMP12 vaccine at 10
5
PFU and two negative control groups receiving an equal
volume of sterile culture L-15 media.
Table 1. Immunization Regimen of CD-1Mice (Dosages Are Calculated in PFU/Mouse)
Group Number of mice
Dosage of 1st
immunization (Day 0)
Dosage of 2nd
immunization (Day 21)
1510
3
(r2segMP12) No injection
2510
4
(r2segMP12) No injection
3510
5
(r2segMP12) No injection
4 5 0 (sterile media; mock control) No injection
5510
5
(parental strain rMP12 control) No injection
6510
3
(r2segMP12) 10
5
(r2segMP12)
7510
4
(r2segMP12) 10
5
(r2segMP12)
8510
5
(r2segMP12) 10
5
(r2segMP12)
9 5 Sterile media; mock control Sterile media; mock control
10 5 10
5
(parental rMP12 strain control) 10
5
(parental rMP12 strain control)
PFU, plaque-forming unit.
FIG. 1. Schematic comparing the RVFV genome with the modified recombinant two-segmented RVFV genome. (a) The
RVFV wild-type genome, which includes the ambisense small (S-) segment consisting of the nucleocapsid (N) protein and
the nonstructural protein, NSs; the negative-sense medium (M-) segment, which contains the structural proteins, Gn and Gc,
and the nonstructural proteins, NSm and 78kD; and the negative-sense large (L-) segment containing the L protein or RNA-
dependent-RNA-polymerase. (b) The modified recombinant bisegmented RVFV genome, which only contains the
S-segment and the L-segment. The NSs coding sequence has been replaced with the Gn and Gc precursors, maintaining the
ambisense coding strategy, and the genome is lacking the authentic M RNA segment. NSm, nonstructural medium; NSs,
nonstructural small; RVFV, Rift Valley fever virus.
IMMUNOGENICITY OF A NOVEL RIFT VALLEY FEVER VIRUS VACCINE 3

Micewereimmunizedsubcutaneouslyonday0ofthe
experiment with an equal volume of their corresponding
immunization (r2segMP12, rMP12, and L-15 media). Be-
fore the initial immunization, all mice were determined to
be healthy and seronegative to RVFV through the analysis
of collected serum using PRNT (data not shown). Animals
were maintained for 6 weeks after the initial immunization.
Mice were immobilized using an isoflurane vaporizer be-
fore blood collection. Whole blood samples of 0.1 mL were
collected from the lateral saphenous vein from immunized
animals at 20 days after initial immunization using a 22g
needle. Serum samples were obtained through centrifuga-
tion of coagulated blood at 2,000 gfor 10 min at 4Cand
used for the detection of neutralizing antibodies using
PRNT. Terminal bleeds were collected at 42 days after
initial immunization by cardiac puncture following iso-
flurane anesthesia and death was confirmed by cervical
dislocation.
Plaque reduction neutralization test
PRNT were used to determine which vaccination regi-
men produced the highest titer of neutralizing antibodies
following the protocol previously described (Roehrig et al,
2008). All serum samples were inactivated at 56C for
30 min and dilutions between 1:10 and 1:320 were tested
(Roehrig et al, 2008). Approximately 50 PFU of the rMP12
vaccine strain was added to each serum concentration and
incubated for 1 h at 37C before infecting the Vero76 cells
in 24-well plates. The wells were then washed with Dul-
becco’s phosphate-buffered saline and overlaid with 1%
methylcellulose.
After 5 days of incubation at 37C, plaques were counted,
and the neutralizing antibody titers were calculated based on
a 50% or greater reduction in plaques from the positive
control (PRNT
50
). Seroconversion was defined using the
cutoff of 1:10 PRNT
50
titer, a seropositive threshold com-
monly used for assessing the neutralizing antibody re-
sponses elicited by arbovirus vaccines ( Julander et al, 2011;
Roehrig et al, 2008; Van Gessel et al, 2011).
Statistical analysis
The PRNT
50
titers of animals receiving each dosage of
the vaccine candidate were compared at 20 days using a
Kruskal–Wallis test followed by Dunnett’s test as the post
hoc multiple comparison procedure, including a compari-
son of each dosage to the parental strain rMP12-positive
control.
Using a Kruskal–Wallis test followed by a Dunnett’s
test post hoc,PRNT
50
titers of animals receiving a single
dose of the r2segMP12 vaccine candidate at varying titers
were compared to mice receiving both an initial dose of
the r2segMP12 vaccine candidate at varying titers and a
booster vaccine at 10
5
PFU at 42 days postimmunization
(dpi). Finally, PRNT
50
titers of animals receiving only a
single dosage of the r2segMP12 vaccine candidate at 10
5
PFU were compared to animals receiving the rMP12
vaccine at 10
5
PFU at 42 dpi with a Mann–Whitney test.
All tests were performed using the GraphPad Prism
(version 8.1.2) program (GraphPad Software, Inc., San
Diego, CA).
Results
Animals from all groups did not show any adverse clin-
ical sign during the experiment. All animals were bled at
20 dpi to evaluate if one single immunization with the
r2segMP12 vaccine candidate elicits neutralizing antibody
responses above the threshold for the correlate of protection
(PRNT
50
>10). All, but one mouse immunized with the
r2segMP12 vaccine candidate seroconverted after a single
immunization (Fig. 2).
Therefore, the r2segMP12 vaccine candidate was capa-
ble of eliciting neutralizing antibody responses in CD-1
mice at dosages between 10
3
and 10
5
PFU. Mice that re-
ceived a single immunization of the r2segMP12 vaccine
candidate at 10
5
PFU produced a significantly higher num-
ber of neutralizing antibodies than mice that received a
single immunization of the r2segMP12 vaccine candidate at
10
3
PFU (Fig. 2, p=0.0139), demonstrating a dose–response
relationship in the vaccine immunogenicity. Importantly,
the comparison of immunogenicity with the rMP12 vac-
cine strain suggests the superior immunogenicity of the
r2segMP12 strain. Mice immunized with r2segMP12 at a
titer of 10
5
PFU had a significantly higher neutralizing an-
tibody response compared to mice that received the rMP12
vaccine at the same titer (Fig. 2, p=0.0079).
To determine if a booster immunization of the r2segMP12
strain can increase immunogenicity and produce long-lasting
neutralizing antibody responses, the PRNT
50
titers were mea-
sured in mice that received varying initial titers of the
r2segMP12, followed by a booster at 21 dpi. Animals in groups
6, 7, and 8 (Table 1) received a booster of the r2segMP12
vaccine candidate at a titer of 10
5
PFU at 21 dpi (Fig. 3).
There was no significant difference in the PRNT
50
titers
between mice that received one single immunization of the
r2segMP12 strain and mice that received a final boost at 21 dpi.
While two mice that received the r2segMP12 vaccine at 10
4
PFU with the addition of the booster had a slightly higher
neutralizing antibody response than mice that received the 10
5
PFU vaccine and booster, there was no significant difference in
the group as a whole. These results demonstrated the immu-
nogenicity of the r2segMP12 strain, with neutralizing antibody
titers suggestive of protection.
Given the observation that a single dose of the
r2segMP12 vaccine candidate at 10
5
PFU elicited a serum
neutralizing antibody response at 42 dpi, the level of anti-
body production was next compared with the neutralizing
antibody response induced by a single dose of the rMP12
vaccine administered at the same titer. Intriguingly, a sin-
gle dose of the r2segMP12 strain produced a significantly
higher titer of neutralizing antibodies than the rMP12 vac-
cine (Fig. 4, *p=0.0238). In addition, serum neutralizing
titers in four out of five mice immunized with the rMP12
vaccine wane below PRNT
50
titer of 10, demonstrating the
need for a booster immunization. These data suggest that the
r2segMP12 strain is superior to the rMP12 vaccine in eli-
citing neutralizing antibody responses in mice.
Collectively, these data suggest that the r2segMP12 strain
is immunogenic and can elicit neutralizing antibody re-
sponses in CD-1 mice that received one single immuniza-
tion. In addition, the lack of the NSs and NSm genes ensures
the safety, but does not compromise the immunogenicity of
the r2segMP12 vaccine strain.
4 AYERS ET AL.

Discussion
Due to the impact of RVFV on both human and livestock
health, efforts to prevent and control RVFV have been
continuous, however, the limitations of each vaccine, mul-
tiple doses required, and expenses to maintain these regi-
mens have made it difficult (Mackenzie, 1935; Pittman et al,
1999; Randall et al, 1962). While live attenuated vaccines
have been developed for RVFV in an effort to eliminate the
need for booster inoculations, several vaccines have dem-
onstrated to be partially attenuated and cause teratogenic
effects and abortions in livestock (Hunter et al, 2002;
Morrill et al, 1997). These disadvantages are problematic,
especially in nonendemic areas during epidemic periods.
There is an urgent need to develop a new vaccine against
RVFV. Therefore, this study sought to establish the immu-
nogenicity of a recombinant RVFV vaccine, containing a two-
segmented viral genome in outbred CD-1 mice. Altogether, the
observations made demonstrated that a single dose of the
r2segMP12 strain induced a neutralizing antibody response in
mice. The neutralizing antibody responses identified in this
study should protect following a challenge with RVFV, al-
though previous studies have shown certain recombinant
vaccines to be protective against lethal RVFV strains with
neutralizing antibody titers as low as 1:4 (Wallace et al, 2006).
The candidate vaccine used in this study was rationally
designed through the deletion of virulence factors, NSs and
NSm, which is based on previous work and because they
have been shown to be virulence factors for wild-type
RVFV (Bird et al, 2011; Ikegami et al, 2006; Won et al,
2007). There is also evidence of the NSs protein contrib-
uting to RVFV disease outcome in mice by modulating host
cell features and defense mechanisms (Leger et al, 2020).
Previously, the generation of viruses lacking the NSs gene
established the product is not essential for replication in mice,
making NSs an accessory protein (Bridgen et al, 2001).
In comparison, the Clone 13 vaccine demonstrated to be
avirulent in mice and highly immunogenic (Muller et al,
1995). However, even with the deletion of the NSs segment
in the Clone 13 strain, it has been reported to cause stillbirths
FIG. 2. Comparison of r2segMP12 and rMP12 neutraliz-
ing antibody response. CD-1 mice (n=10 per group) were
administered either r2segMP12 stain at one of three titers
(10
3
,10
4
,or10
5
PFU) or rMP12 strain (10
5
PFU). Serum
was collected at 20 dpi and antibody titer was measured by
PRNT
50
with a 1:10 neutralizing antibody titer used as the
threshold for the correlate of protection (dotted line). The
Kruskal–Wallis test with Dunnett’s post hoc multiple com-
parison test was used. ‘‘*’’ Indicates the significant differ-
ence identified by the Kruskal–Wallis test plus Dunnett’s
post hoc test ( p=0.0151). ‘‘**’’ Indicated the significant
difference identified by the Kruskal–Wallis test plus Dun-
nett’s post hoc test ( p=0.0087). The bar lines represent the
medians of values from that group of animals. PFU, plaque-
forming units; PRNT, plaque reduction neutralization tests.
FIG. 3. Comparison of PRNT
50
titers from different doses
of the r2segMP12 vaccine candidate on 42dpi. CD-1 mice
(n=5 per group) were administered either a single dose of
r2segMP12 (at either 10
3
[filled circles], 10
4
[filled squares],
or 10
5
[filled triangles] PFU), or two doses (single dose and
a booster dose) at the same titer (primary immunization at
either 10
3
,10
4
,or10
5
PFU; booster at 10
5
PFU). Serum
neutralizing activity was measured by PRNT
50
and com-
pared by Kruskal–Wallis test followed by a Dunnett’s test as
the post hoc multiple comparison procedure. The bars rep-
resent the median and the threshold of protection is marked
by the dotted line.
FIG. 4. Comparison of PRNT
50
titers between mice re-
ceiving one single immunization with the rMP12 strain
and the r2segMP12 strain at 42 days after immunization. CD-
1mice(n=5 per group) were administered a single dose of
either r2segMP12 or rMP12 at 10
5
PFU/mL and antibody titer
was measured by PRNT
50
and compared by Mann–Whitney
test with the bars representing the median. *Indicates p<0.05.
IMMUNOGENICITY OF A NOVEL RIFT VALLEY FEVER VIRUS VACCINE 5

and fetal infections when administered in an overdose to
pregnant ewes in their first trimester (Makoschey et al, 2016).
While other RVFV candidate live attenuated vaccines
have been developed through the deliberate deletion of NSs
and NSm genes and demonstrated to be safe and immu-
nogenic in mice and pregnant sheep (Bird et al, 2011; Bird
et al, 2008), our work has important implications for the
development of RVFV candidate live attenuated vaccines.
While these vaccines were made using similar methods, the
r2segMP12 strain with a two-segmented genome will have a
reduced likelihood for reversion to the virulent phenotype.
In addition, the r2segMP12 strain proves that the double
deletion of NSs and NSm genes does not cause reduced
immunogenicity relative to the rMP12 strain (Brennan et al,
2011).
The results of this study also determined that the r2segMP12
strain elicited a significantly higher level of neutralizing
antibody response than the conditionally licensed rMP12
vaccine at 20 and 42 dpi. In addition, the r2segMP12 strain
does not express the NSs and NSm proteins, providing the
basis for differentiating infected from vaccinated animals
(DIVA) (McElroy et al, 2009). Specifically, the lack of anti-
NSs antibodies has been developed as a DIVA marker for
serological diagnosis. Hence, the r2segMP12 strain has the
potential utility for the deployment of rapid outbreak re-
sponses.
We conclude that the superior immunogenicity of the
r2segMP12 strain warrants its advancement in the process of
vaccine development, including challenge protection studies
conducted in mice and then sheep, which are the amplify-
ing hosts for RVFV. Future experiments will focus on
the characterization of the immune response induced by
r2segMP12 and its ability to protect against a lethal RVFV
challenge.
Acknowledgments
The authors thank Susan M. Hettenbach for her admin-
istrative support. This work was, in part, based on research
performed for Victoria Ayers’s doctoral dissertation (avail-
able at: https://krex.k-state.edu/dspace/bitstream/handle/2097/
42420/VictoriaAyers2022.pdf?isAllowed=y&sequence=1).
Authors’ Contributions
V.B.A.: investigation and writing—original draft, Y-.J.S.H.:
investigation and writing—review and editing, J.I.D.: vali-
dation, resources, and writing—review and editing, A.K.:
validation, resources, and writing—review and editing,
B.B.: validation, resources, and writing—review and edit-
ing, S.H.: conceptualization, funding acquisition, and writing—
review and editing, D.L.V.: conceptualization, funding ac-
quisition, and writing—review and editing.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
This work was supported by the National Institute of
Food and Agriculture, U.S. Department of Agriculture
(award number 2015-67015-22961) (SH) and by the UK
Biotechnology and Biological Sciences Research Council
(award number BB/M027112/1) (AK) as part of the joint
NIFA-BBSRC Animal Health and Disease program. The
work was also supported by the UK Medical Research
Council (MC_UU_12014/8) (AK). The funders had no role
in study design, data collection and analysis, decision to
publish, or preparation of the article.
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Address correspondence to:
Dr. Dana L. Vanlandingham
Department of Diagnostic Medicine/Pathobiology
College of Veterinary Medicine
Kansas State University
Manhattan, KS 66506
USA
E-mail: dlvanlan@vet.k-state.edu
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