Serosurveillance for Severe Acute Respiratory Syndrome Coronavirus 2 Antibody in Feral Swine and White-Tailed Deer in Texas.

Background: Serological evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been reported in white-tailed deer (WTD) in the United States and Canada. Even though WTD are susceptible to SARS-CoV-2 infection, there is no evidence of infection by this virus in other mammalian species that might interact with WTD in nature. Similar to WTD, feral swine are widely distributed and generally occupy the same range as WTD in Texas. The objective of this study was to determine the prevalence of SARS-CoV-2 neutralizing antibody in WTD during 2020 and 2021 and determine the prevalence of SARS-CoV-2 neutralizing antibody in feral swine during 2018 (prepandemic period) and from March 2020 to February 2021 (pandemic period) in Travis County, Texas. Materials and Methods: Sera samples were collected from hunter-killed WTD and feral swine during the prepandemic and pandemic period and tested for SARS-CoV-2 antibody by a plaque reduction neutralization assay in Vero cells. Results: SARS-CoV-2 antibody was not detected in any of the 166 feral swine sera samples, including 24 samples collected during the prepandemic and 142 samples collected during the pandemic period. Furthermore, SARS-CoV-2 antibody was not detected in the 115 WTD samples collected during late 2020, but antibody was detected in WTD in early 2021. Conclusions: The results indicated that SARS-CoV-2 infection of WTD occurred during early 2021 in Travis County, Texas, but serological evidence of SARS-CoV-2 infection was not detected in the feral swine samples collected from the same locality and during the same time period of the collection of WTD samples.

Estimation of the Minimal Rift Valley Fever Virus Protective Neutralizing Antibody Titer in Human Volunteers Immunized with MP-12 Vaccine Based on Protection in a Mouse Model of Disease.

The Rift Valley fever virus (RVFV) MP-12 vaccine is a promising human and veterinary vaccine. Although the vaccine elicited neutralizing antibody (nAb) in human volunteers, the minimal antibody titer that is needed to afford protection is unknown. Therefore, this study was conducted to determine the minimal nAb titer elicited by the RVFV MP-12 vaccine in human volunteers that protected mice against lethal RVFV challenge as a surrogate assessment of the protective efficacy of the vaccine. Among volunteers who were vaccinated with the MP-12 vaccine during a phase II trial, sera with antibody titers of 1:20 collected 5 years post-vaccination (PV), 1:40 titer collected 2 years PV, and 1:80 titer collected 1 year PV was passively transferred to groups of BALB/c mice. Blood samples were obtained 1 day after passive transfer to determine the RVFV neutralizing nAb titer before challenge with pathogenic RVFV (strain ZH501). Our results indicated that 1 day after passive transfer of the immune sera, an approximate 4-fold reduction in circulating nAb titers was detected in the mice. The presence of RVFV nAb titers in the range of 1:5 to 1:20 were generally protective (75-100% survival). These results suggested that circulating titers of 1:5 or higher offer a high degree of protection by MP-12-elicited antibody in human volunteers. Also, the findings highlighted the value of using the BALB/c mouse RVFV challenge model as a surrogate for evaluating the protective nAb responses elicited by MP-12 and possible use for evaluating the efficacy of other RVFV vaccine candidates.

SARS-CoV-2 Neutralizing Antibodies in White-Tailed Deer from Texas.

Serological evidence of SARS-CoV-2 infection among white-tailed deer has been reported from Illinois, Michigan, Pennsylvania, and New York. This study was conducted to determine whether deer in Texas also had evidence of SARS-CoV-2 infection. Archived sera samples collected from deer in Travis County, Texas, during 2018, before and during the pandemic in 2021 were tested for neutralizing antibody to this virus by a standard plaque reduction neutralization assay. SARS-CoV-2 antibody was not detected in 40 deer sera samples collected during 2018, but 37% (20/54) samples collected in 2021 were positive for antibody. The seroprevalence rate between males and females differed significantly (p < 0.05) and the highest rate (82%) was detected in the 1.5-year-old animals. These findings extended the geographical range of prior SARS-CoV-2 infection among white-tailed deer in the United States and further confirm that infection was common among this species.

Serological Evidence of West Nile Virus Infection in White-Tailed Deer in Central Texas.

White-tailed deer (WTD) are abundant mammals widely distributed across the United States. As a result, WTD are considered to be excellent sentinels for detecting arboviral activity in certain geographic areas. Evidence of West Nile virus (WNV) antibody in WTD has been reported previously in several states. However, WNV infection in WTD has not been reported from Texas, where the incidence of human West Nile (WN) cases is among the highest in the United States. Therefore, the aim of this study was to determine the prevalence of WNV antibody in WTD in central Texas. Sera samples (n = 644) were collected from deer during the fall and winter in western Travis County, Texas from 2014 to 2018 and tested for WNV immunoglobulin G (IgG) antibody by an indirect enzyme-linked immunosorbent assay (ELISA). ELISA antibody-positive samples were further tested for WNV and St. Louis encephalitis virus (SLEV) antibodies by an 80% plaque-reduction neutralization tests (PRNT80). Overall, 9% (n = 58) and 0.31% (n = 2) of the deer samples had serological evidence of WNV and SLEV infections, respectively. WNV seroprevalence differed significantly by age (p < 0.05), but there was no significant difference between sex. Interestingly, 3.1% (n = 20) of the samples were positive for Flavivirus IgG antibody by ELISA, but negative for SLEV and WNV antibodies, suggesting that other Flaviviruses may be circulating among WTD in Texas. Finally, these results supported WNV infection among WTD and highlight their potential role as sentinels for the detection of WNV in Texas and warrant further studies to determine the role WTD play in the maintenance and transmission of WNV.

Theoretical risk of genetic reassortment should not impede development of live, attenuated Rift Valley fever (RVF) vaccines commentary on the draft WHO RVF Target Product Profile.

In November 2019, The World Health Organization (WHO) issued a draft set of Target Product Profiles (TPPs) describing optimal and minimally acceptable targets for vaccines against Rift Valley fever (RVF), a Phlebovirus with a three segmented genome, in both humans and ruminants. The TPPs contained rigid requirements to protect against genomic reassortment of live, attenuated vaccines (LAVs) with wild-type RVF virus (RVFV), which place undue constraints on development and regulatory approval of LAVs. We review the current LAVs in use and in development, and conclude that there is no evidence that reassortment between LAVs and wild-type RVFV has occurred during field use, that such a reassortment event if it occurred would have no untoward consequence, and that the TPPs should be revised to provide a more balanced assessment of the benefits versus the theoretical risks of reassortment.

Safety and immunogenicity of Rift Valley fever MP-12 and arMP-12ΔNSm21/384 vaccine candidates in goats (Capra aegagrus hircus) from Tanzania.

Vaccination of domestic ruminants is considered to be an effective strategy for protecting these animals against Rift Valley fever (RVF), but available vaccines have limitations. Therefore, the aim of this study was to determine the safety and immunogenicity of RVF virus (RVFV) mutagenesis passage 12 (MP-12) and arMP-12ΔNSm21/384 vaccine candidates in goats (Capra aegagrus hircus) in Tanzania. Goats were vaccinated intramuscularly with RVFV MP-12 or arMP-12ΔNSm21/384, and then on Day 87 post-vaccination (PV) all animals were revaccinated using the RVFV MP-12 vaccine candidate. Serum samples were collected from the animals before and after vaccination at various intervals to test for RVFV using a Vero cell culture assay and reverse transcription polymerase chain reaction and for RVFV-neutralising antibody using a plaque reduction neutralisation assay. Serum samples collected before vaccination on Days -14 and 0, and on Days 3, 4 and 5 PV were negative for RVFV and neutralising antibody. All animals remained healthy, and viremia was not detected in any of the animals. Rift Valley fever virus antibody was first detected on Day 5 PV at a 1:10 dilution in five of five animals vaccinated with the MP-12 vaccine and in five of eight animals vaccinated with arMP-12ΔNSm21/384. Titres then increased and were sustained at 1:40 to 1:640 through to Day 87 PV. All animals that were revaccinated on Day 87 PV with MP-12 developed antibody titres ranging from 1:160 to as high as 1:10 240 on Days 14 and 21 PV. Although the antibody titres for goats vaccinated with RVF MP-12 were slightly higher than titres elicited by the arMP-12ΔNSm21/384 vaccine, these findings demonstrated that both vaccines are promising candidates for the prevention of RVF among Tansanian goats.

Distinct virulence of Rift Valley fever phlebovirus strains from different genetic lineages in a mouse model.

Rift Valley fever phlebovirus (RVFV) causes high rates of abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral transmission occurs via mosquito vectors in endemic areas, which necessitates regular vaccination of susceptible livestock animals to prevent the RVF outbreaks. Although ZH501 strain has been used as a challenge strain for past vaccine efficacy studies, further characterization of other RVFV strains is important to optimize ruminant and nonhuman primate RVFV challenge models. This study aimed to characterize the virulence of wild-type RVFV strains belonging to different genetic lineages in outbred CD1 mice. Mice were intraperitoneally infected with 1x103 PFU of wild-type ZH501, Kenya 9800523, Kenya 90058, Saudi Arabia 200010911, OS1, OS7, SA75, Entebbe, or SA51 strains. Among them, mice infected with SA51, Entebbe, or OS7 strain showed rapid dissemination of virus in livers and peracute necrotic hepatitis at 2-3 dpi. Recombinant SA51 (rSA51) and Zinga (rZinga) strains were recovered by reverse genetics, and their virulence was also tested in CD1 mice. The rSA51 strain reproduced peracute RVF disease in mice, whereas the rZinga strain showed a similar virulence with that of rZH501 strain. This study showed that RVFV strains in different genetic lineages display distinct virulence in outbred mice. Importantly, since wild-type RVFV strains contain defective-interfering RNA or various genetic subpopulations during passage from original viral isolations, recombinant RVFV strains generated by reverse genetics will be better suitable for reproducible challenge studies for vaccine development as well as pathological studies.

Seroprevalence of Borrelia burgdorferi antibodies in white-tailed deer from Texas.

Lyme Disease is caused by the bacterial pathogen Borrelia burgdorferi, and is transmitted by the tick-vector Ixodes scapularis. It is the most prevalent arthropod-borne disease in the United States. To determine the seroprevalence of B. burgdorferi antibodies in white-tailed deer (Odocoileus virginianus) from Texas, we analyzed serum samples (n = 1493) collected during the 2001-2015 hunting seasons, using indirect ELISA. Samples with higher sero-reactivity (0.803 and above) than the negative control group (0.662) were further tested using a more specific standardized western immunoblot assay to rule out false positives. Using ELISA, 4.7% of the samples were sero-reactive against B. burgdorferi, and these originated in two eco-regions in Texas (Edwards Plateau and South Texas Plains). However, only 0.5% of the total samples were sero-reactive by standardized western immunoblot assay. Additionally, both ELISA and standardized western immunoblot assay results correlated with an increased incidence in human Lyme Disease cases reported in Texas. This is the first longitudinal study to demonstrate fluctuation in sero-reactivity of white-tailed deer to B. burgdorferi sensu stricto antigens in southern United States. Future ecological and geographical studies are needed to assess the environmental factors governing the prevalence of Lyme Disease in non-endemic areas of the southern United States.

Correction: Correlative Gene Expression to Protective Seroconversion In Rift Valley Vaccinates.

[This corrects the article DOI: 10.1371/journal.pone.0147027.].

Correlative Gene Expression to Protective Seroconversion in Rift Valley Fever Vaccinates.

Rift Valley fever Virus (RVFV), a negative-stranded RNA virus, is the etiological agent of the vector-borne zoonotic disease, Rift Valley fever (RVF). In both humans and livestock, protective immunity can be achieved through vaccination. Earlier and more recent vaccine trials in cattle and sheep demonstrated a strong neutralizing antibody and total IgG response induced by the RVF vaccine, authentic recombinant MP-12 (arMP-12). From previous work, protective immunity in sheep and cattle vaccinates normally occurs from 7 to 21 days after inoculation with arMP-12. While the serology and protective response induced by arMP-12 has been studied, little attention has been paid to the underlying molecular and genetic events occurring prior to the serologic immune response. To address this, we isolated RNA from whole blood of vaccinated calves over a time course of 21 days before and after vaccination with arMP-12. The time course RNAs were sequenced by RNASeq and bioinformatically analyzed. Our results revealed time-dependent activation or repression of numerous gene ontologies and pathways related to the vaccine induced immune response and its regulation. Additional bioinformatic analyses identified a correlative relationship between specific host immune response genes and protective immunity prior to the detection of protective serum neutralizing antibody responses. These results contribute an important proof of concept for identifying molecular and genetic components underlying the immune response to RVF vaccination and protection prior to serologic detection.

Rift Valley fever MP-12 vaccine Phase 2 clinical trial: Safety, immunogenicity, and genetic characterization of virus isolates.

An outbreak or deliberate release of Rift Valley fever (RVF) virus could have serious public health and socioeconomic consequences. A safe RVF vaccine capable of eliciting long-lasting immunity after a single injection is urgently needed. The live attenuated RVF MP-12 vaccine candidate has shown promise in Phase 1 clinical trials; no evidence of reversion to virulence has been identified in numerous animal studies. The objective of this Phase 2 clinical trial was to (a) further examine the safety and immunogenicity of RVF MP-12 in RVF virus-naïve humans and (b) characterize isolates of RVF MP-12 virus recovered from the blood of vaccinated subjects to evaluate the genetic stability of MP-12 attenuation. We found that RVF MP-12 was well tolerated, causing mostly mild reactions that resolved without sequelae. Of 19 subjects, 18 (95%) and 19 (100%) achieved, respectively, 80% and 50% plaque reduction neutralization titers (PRNT80 and PRNT50)≥1:20 by postvaccination day 28. All 18 PRNT80 responders maintained PRNT80 and PRNT50≥1:40 until at least postvaccination month 12. Viremia was undetectable in the plasma of any subject by direct plaque assay techniques. However, 5 of 19 vaccinees were positive for MP-12 isolates in plasma by blind passage of plasma on Vero cells. Vaccine virus was also recovered from buffy coat material from one of those vaccinees and from one additional vaccinee. Through RNA sequencing of MP-12 isolates, we found no reversions of amino acids to those of the parent virulent virus (strain ZH548). Five years after a single dose of RVF MP-12 vaccine, 8 of 9 vaccinees (89%) maintained a PRNT80≥1:20. These findings support the continued development of RVF MP-12 as a countermeasure against RVF virus in humans.

Mapping a Major Gene for Resistance to Rift Valley Fever Virus in Laboratory Rats.

The Rift Valley Fever virus (RVFV) presents an epidemic and epizootic threat in sub-Saharan Africa, Egypt, and the Arabian Peninsula, and has furthermore recently gained attention as a potential weapon of bioterrorism due to its ability to infect both livestock and humans. Inbred rat strains show similar characteristic responses to the disease as humans and livestock, making them a suitable model species. Previous studies had indicated differences in susceptibility to RVFV hepatic disease among various rat strains, including a higher susceptibility of Wistar-Furth (WF) compared to a more resistant Lewis (LEW) strain. Further study revealed that this resistance trait exhibits the pattern of a major dominant gene inherited in Mendelian fashion. A genome scan of a congenic WF.LEW strain, created from the susceptible WF and resistant LEW strains and itself resistant to infection with RVFV, revealed 2 potential regions for the location of the gene, 1 on chromosome 3 and the other on chromosome 9. Through backcrossing of WF.LEW rats to WF rats, genotyping offspring using SNPs and microsatellites, and viral challenges of 3 N1 litters, we have mapped the gene to the distal end of chromosome 3.

Development of a novel, single-cycle replicable rift valley Fever vaccine.

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is an arbovirus that causes severe disease in humans and livestock in sub-Saharan African countries. Although the MP-12 strain of RVFV is a live attenuated vaccine candidate, neuroinvasiveness and neurovirulence of MP-12 in mice may be a concern when vaccinating certain individuals, especially those that are immunocompromised. We have developed a novel, single-cycle replicable MP-12 (scMP-12), which carries an L RNA, M RNA mutant encoding a mutant envelope protein lacking an endoplasmic reticulum retrieval signal and defective for membrane fusion function, and S RNA encoding N protein and green fluorescent protein. The scMP-12 underwent efficient amplification, then formed plaques and retained the introduced mutation after serial passages in a cell line stably expressing viral envelope proteins. However, inoculation of the scMP-12 into naïve cells resulted in a single round of viral replication, and production of low levels of noninfectious virus-like particles. Intracranial inoculation of scMP-12 into suckling mice did not cause clinical signs or death, a finding which demonstrated that the scMP-12 lacked neurovirulence. Mice immunized with a single dose of scMP-12 produced neutralizing antibodies, whose titers were higher than in mice immunized with replicon particles carrying L RNA and S RNA encoding N protein and green fluorescent protein. Moreover, 90% of the scMP-12-immunized mice were protected from wild-type RVFV challenge by efficiently suppressing viremia and replication of the challenge virus in the liver and the spleen. These data demonstrated that scMP-12 is a safe and immunogenic RVFV vaccine candidate.

Efficacy of a recombinant Rift Valley fever virus MP-12 with NSm deletion as a vaccine candidate in sheep.

Rift Valley fever virus (RVFV), a mosquito-borne virus in the Bunyaviridae family and Phlebovirus genus, causes RVF, a disease of ruminants and man, endemic in Sub-Saharan African countries. However, outbreaks in Yemen and Saudi Arabia demonstrate the ability for RVFV to spread into virgin territory and thus the need exists to develop safe and efficacious vaccines that can be used outside the endemic zones. Commercial RVFV vaccines are available but have limitations that prevent their use in disease-free countries. Consequently, there are ongoing efforts to develop and/or improve RVFV vaccines with global acceptability. In this study a previously developed MP-12-derived vaccine candidate with a large deletion of the NSm gene in the pre Gn region of the M segment (arMP-12-ΔNSm21/384) developed by T. Ikegami, that was already shown to be safe in pregnant sheep causing neither abortion nor fetal malformation was further evaluated. This vaccine was tested for protection of sheep from viremia and fever following challenge with virulent RVFV ZH501 strain. A single vaccination with arMP-12-ΔNSm21/384 fully protected sheep when challenged four weeks post vaccination, thereby demonstrating that this vaccine is efficacious in protecting these animals from RVFV infection.

Immunogenicity of a recombinant Rift Valley fever MP-12-NSm deletion vaccine candidate in calves.

The safety and immunogenicity of an authentic recombinant (ar) of the live, attenuated MP-12 Rift Valley fever (RVF) vaccine virus with a large deletion of the NSm gene in the pre-Gn region of the M RNA segment (arMP-12ΔNSm21/384) was tested in 4-6 month old Bos taurus calves. Phase I of this study evaluated the neutralizing antibody response, measured by 80% plaque reduction neutralization (PRNT80), and clinical response of calves to doses of 1 × 10(1) through 1 × 10(7) plaque forming units (PFU) administered subcutaneously (s.c.). Phase II evaluated the clinical and neutralizing antibody response of calves inoculated s.c. or intramuscularly (i.m.) with 1 × 10(3), 1 × 10(4) or 1 × 10(5)PFU of arMP-12ΔNSm21/384. No significant adverse clinical events were observed in the animals in these studies. Of all specimens tested, only one vaccine viral isolate was recovered and that virus retained the introduced deletion. In the Phase I study, there was no statistically significant difference in the PRNT80 response between the dosage groups though the difference in IgG response between the 1 × 10(1)PFU group and the 1 × 10(5)PFU group was statistically significant (p<0.05). The PRNT80 response of the respective dosage groups corresponded to dose of vaccine with the 1 × 10(1)PFU dose group showing the least response. The Phase II study also showed no statistically significant difference in PRNT80 response between the dosage groups though the difference in RVFV-specific IgG values was significantly increased (p<0.001) in animals inoculated i.m. with 1 × 10(4) or 1 × 10(5)PFU versus those inoculated s.c. with 1 × 10(3) or 1 × 10(5)PFU. Although the study groups were small, these data suggest that 1 × 10(4) or 1 × 10(5)PFU of arMP-12ΔNSm21/384 administered i.m. to calves will consistently stimulate a presumably protective PRNT80 response for at least 91 days post inoculation. Further studies of arMP-12ΔNSm21/384 are warranted to explore its suitability as an efficacious livestock vaccine.

The nucleocapsid protein of Rift Valley fever virus is a potent human CD8+ T cell antigen and elicits memory responses.

There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8(+) T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8(+) T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N(121-129)) and IL9 (ILDAHSLYL, N165-173), were defined. VT9- and IL9-specific CD8(+) T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8(+) T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8(+) T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8(+) T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.

Safety and immunogenicity of recombinant Rift Valley fever MP-12 vaccine candidates in sheep.

The safety and immunogenicity of two authentic recombinant (ar) Rift Valley fever (RVF) viruses, one with a deletion in the NSs region of the S RNA segment (arMP-12ΔNSs16/198) and the other with a large deletion of the NSm gene in the pre Gn region of the M RNA segment (arMP-12ΔNSm21/384) of the RVF MP-12 vaccine virus were tested in crossbred ewes at 30-50 days of gestation. First, we evaluated the neutralizing antibody response, measured by plaque reduction neutralization (PRNT(80)), and clinical response of the two viruses in groups of four ewes each. The virus dose was 1×10(5)plaque forming units (PFU). Control groups of four ewes each were also inoculated with a similar dose of RVF MP-12 or the parent recombinant virus (arMP-12). Neutralizing antibody was first detected in 3 of 4 animals inoculated with arMP-12ΔNSm21/384 on Day 5 post inoculation and all four animals had PRNT(80) titers of ≥1:20 on Day 6. Neutralizing antibody was first detected in 2 of 4 ewes inoculated with arMP-12ΔNSs16/198 on Day 7 and all had PRNT(80) titers of ≥1:20 on Day 10. We found the mean PRNT(80) response to arMP-12ΔNSs16/198 to be 16- to 25-fold lower than that of ewes inoculated with arMP-12ΔNSm21/384, arMP-12 or RVF MP-12. No abortions occurred though a single fetal death in each of the arMP-12 and RVF MP-12 groups was found at necropsy. The poor PRNT(80) response to arMP-12ΔNSs16/198 caused us to discontinue further testing of this candidate and focus on arMP-12ΔNSm21/384. A dose escalation study of arMP-12ΔNSm21/384 showed that 1×10(3)plaque forming units (PFU) stimulate a PRNT(80) response comparable to doses of up to 1×10(5)PFU of this virus. With further study, the arMP-12ΔNSm21/384 virus may prove to be a safe and efficacious candidate for a livestock vaccine. The large deletion in the NSm gene may also provide a negative marker that will allow serologic differentiation of naturally infected animals from vaccinated animals.

Genetic subpopulations of Rift Valley fever virus strains ZH548 and MP-12 and recombinant MP-12 strains.

Rift Valley fever virus strain MP-12 was generated by serial plaque passages of parental strain ZH548 12 times in MRC-5 cells in the presence of a chemical mutagen, 5-fluorouracil. As a result, MP-12 encoded 4, 9, and 10 mutations in the S, M, and L segments, respectively. Among them, mutations in the M and L segments were responsible for attenuation, while the MP-12 S segment still encoded a virulent phenotype. We performed high-throughput sequencing of MP-12 vaccine, ZH548, and recombinant MP-12 (rMP-12) viruses. We found that rMP-12 contains very low numbers of viral subpopulations, while MP-12 and ZH548 contain 2 to 4 times more viral genetic subpopulations than rMP-12. MP-12 genetic subpopulations did not encode the ZH548 sequence at the 23 MP-12 consensus mutations. On the other hand, 4 and 2 mutations in M and L segments of MP-12 were found in ZH548 subpopulations. Thus, those 6 mutations were no longer MP-12-specific mutations. ZH548 encoded several unique mutations compared to other Egyptian strains, i.e., strains ZH501, ZH1776, and ZS6365. ZH548 subpopulations shared nucleotides at the mutation site common with those in the Egyptian strains, while MP-12 subpopulations did not share those nucleotides. Thus, MP-12 retains unique genetic subpopulations and has no evidence of reversion to the ZH548 sequence in the subpopulations. This study provides the first information regarding the genetic subpopulations of RVFV and shows the genetic stability of the MP-12 vaccine manufactured in MRC-5 cells.

Mucosal immunization of rhesus macaques with Rift Valley Fever MP-12 vaccine.

Rhesus macaques given 5 × 10(4) or 1 × 10(5) plaque-forming units (pfu) of Rift Valley fever (RVF) MP-12 vaccine by oral, intranasal drops, or small particle aerosol showed no adverse effects up to 56 days after administration. All monkeys given the vaccine by aerosol or intranasal drops developed 80% plaque reduction neutralization titers of ≥ 1:40 by day 21 after inoculation. Only 2 of 4 monkeys given the vaccine by oral instillation developed detectable neutralizing antibodies. All monkeys vaccinated by mucosal routes that developed detectable neutralizing antibodies were protected against viremia when challenged with 1 × 10(5) pfu of virulent RVF virus delivered by a small particle aerosol at 56 days after vaccination. A single inoculation of the RVF MP-12 live attenuated vaccine by the aerosol or intranasal route may provide an alternative route of protective immunization to RVFV in addition to conventional intramuscular injection.

Protection of MP-12-vaccinated rhesus macaques against parenteral and aerosol challenge with virulent rift valley fever virus.

To test safety and efficacy of the Rift Valley fever MP-12 (RVF MP-12) vaccine, 9 healthy adult Rhesus macaques, weighing 5-10 kg, were inoculated intramuscularly with 6 × 10(3) plaque forming units (PFUs) of MP-12 vaccine. The monkeys developed neutralizing antibody responses with no adverse effects other than a transient, low-titer viremia in 3 monkeys. Four vaccinated animals challenged intravenously with 3 × 10(6) PFUs of virulent Rift Valley fever virus strain ZH-501 (RVFV ZH-501) at 126 days after vaccination were protected against infection. The remaining 5 vaccinated monkeys along with 2 monkeys that had been vaccinated 6 years prior were completely protected against a small particle aerosol challenge of 5 × 10(5) PFUs of RVFV ZH-501. The mutagen-attenuated RVF MP-12 vaccine was determined to be protective against intravenous and aerosol challenge with virulent RVFV in these macaques, which suggests further development as a vaccine for humans is warranted.