Safety and Efficacy upon Infection in Sheep with Rift Valley Fever Virus ZH548-rA2, a Triple Mutant Rescued Virus.

The introduction of three single nucleotide mutations into the genome of the virulent RVFV ZH548 strain allows for the rescue of a fully attenuated virus in mice (ZH548-rA2). These mutations are located in the viral genes encoding the RdRp and the non-structural protein NSs. This paper shows the results obtained after the subcutaneous inoculation of ZH548-rA2 in adult sheep and the subsequent challenge with the parental virus (ZH548-rC1). Inoculation with the ZH548-rA2 virus caused no detectable clinical or pathological effect in sheep, whereas inoculation of the parental rC1 virus caused lesions compatible with viral infection characterised by the presence of scattered hepatic necrosis. Viral infection was confirmed via immunohistochemistry, with hepatocytes within the necrotic foci appearing as the main cells immunolabelled against viral antigen. Furthermore, the inoculation of sheep with the rA2 virus prevented the liver damage expected after rC1 virus inoculation, suggesting a protective efficacy in sheep which correlated with the induction of both humoral and cell-mediated immune responses.

Identification of Single Amino Acid Changes in the Rift Valley Fever Virus Polymerase Core Domain Contributing to Virus Attenuation In Vivo.

Rift Valley fever (RVF) is an arboviral zoonotic disease affecting many African countries with the potential to spread to other geographical areas. RVF affects sheep, goats, cattle and camels, causing a high rate of abortions and death of newborn lambs. Also, humans can be infected, developing a usually self-limiting disease that can turn into a more severe illness in a low percentage of cases. Although different veterinary vaccines are available in endemic areas in Africa, to date no human vaccine has been licensed. In previous works, we described the selection and characterization of a favipiravir-mutagenized RVFV variant, termed 40Fp8, with potential as a RVF vaccine candidate due to the strong attenuation shown in immunocompromised animal models. Compared to the parental South African 56/74 viral strain, 40Fp8 displayed 7 amino acid substitutions in the L-protein, three of them located in the central region corresponding to the catalytic core of the RNA-dependent RNA polymerase (RdRp). In this work, by means of a reverse genetics system, we have analyzed the effect on virulence of these amino acid changes, alone or combined, both in vitro and in vivo. We found that the simultaneous introduction of two changes (G924S and A1303T) in the heterologous ZH548-RVFV Egyptian strain conferred attenuated phenotypes to the rescued viruses as shown in infected mice without affecting virus immunogenicity. Our results suggest that both changes induce resistance to favipiravir likely associated to some fitness cost that could be the basis for the observed attenuation in vivo. Conversely, the third change, I1050V, appears to be a compensatory mutation increasing viral fitness. Altogether, these results provide relevant information for the safety improvement of novel live attenuated RVFV vaccines.

Use of Foot-and-Mouth Disease Virus Non-coding Synthetic RNAs as Vaccine Adjuvants.

The ncRNAs are short RNA transcripts with sequence and structure resembling that of specific domains in the non-coding regions of the foot-and-mouth disease (FMD) virus (FMDV ) genome. These synthetic molecules induce a robust antiviral response and have been shown to enhance the immune response and protection induced by an FMD inactivated vaccine in pigs. Here, we describe the method for ncRNAs synthesis, formulation, and delivery into mice and pigs for studies focused on testing the adjuvant effect of RNA-based strategies in combination with veterinarian vaccines.

Using RVFV as a Vector Platform for the Expression of Ruminant Disease Antigens.

Live attenuated viruses remain as vaccine agents with unparalleled performance in terms of duration, magnitude, and breadth of induced immune responses. As the yellow fever-attenuated vaccine strain Y17D, attenuated Rift Valley fever virus shares features suitable to be used as a viral vector for heterologous antigen expression and bivalent vaccine development. Current reverse genetics technology showed the successful rescue of RVFV carrying foreign antigens with little immunogenicity loss in experimental animal models. We show here the basic experimental protocol to achieve the expression of candidate vaccine antigens from other important diseases of ruminants using RVFV as a vector platform as well as preliminary steps for the characterization of immunogenicity in vivo.

Evaluation of silver nanoparticles for the prevention of SARS-CoV-2 infection in health workers: In vitro and in vivo.

SARS-CoV-2 infection in hospital areas is of a particular concern, since the close interaction between health care personnel and patients diagnosed with COVID-19, which allows virus to be easily spread between them and subsequently to their families and communities. Preventing SARS-CoV-2 infection among healthcare personnel is essential to reduce the frequency of infections and outbreaks during the pandemic considering that they work in high-risk areas. In this research, silver nanoparticles (AgNPs) were tested in vitro and shown to have an inhibitory effect on SARS-CoV-2 infection in cultured cells. Subsequently, we assess the effects of mouthwash and nose rinse with ARGOVIT® silver nanoparticles (AgNPs), in the prevention of SARS-CoV-2 contagion in health workers consider as high-risk group of acquiring the infection in the General Tijuana Hospital, Mexico, a hospital for the exclusive recruitment of patients diagnosed with COVID-19. We present a prospective randomized study of 231 participants that was carried out for 9 weeks (during the declaration of a pandemic). The "experimental" group was instructed to do mouthwash and nose rinse with the AgNPs solution; the "control" group was instructed to do mouthwashes and nose rinse in a conventional way. The incidence of SARS-CoV-2 infection was significantly lower in the "experimental" group (two participants of 114, 1.8%) compared to the "control" group (thirty-three participants of 117, 28.2%), with an 84.8% efficiency. We conclude that the mouth and nasal rinse with AgNPs helps in the prevention of SARS-CoV-2 infection in health personnel who are exposed to patients diagnosed with COVID-19.

The Change P82L in the Rift Valley Fever Virus NSs Protein Confers Attenuation in Mice.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes an important disease in ruminants, with great economic losses. The infection can be also transmitted to humans; therefore, it is considered a major threat to both human and animal health. In a previous work, we described a novel RVFV variant selected in cell culture in the presence of the antiviral agent favipiravir that was highly attenuated in vivo. This variant displayed 24 amino acid substitutions in different viral proteins when compared to its parental viral strain, two of them located in the NSs protein that is known to be the major virulence factor of RVFV. By means of a reverse genetics system, in this work we have analyzed the effect that one of these substitutions, P82L, has in viral attenuation in vivo. Rescued viruses carrying this single amino acid change were clearly attenuated in BALB/c mice while their growth in an interferon (IFN)-competent cell line as well as the production of interferon beta (IFN-ß) did not seem to be affected. However, the pattern of nuclear NSs accumulation was modified in cells infected with the mutant viruses. These results highlight the key role of the NSs protein in the modulation of viral infectivity.

Precise location of linear epitopes on the capsid surface of feline calicivirus recognized by neutralizing and non-neutralizing monoclonal antibodies.

We report the generation, characterization and epitope mapping of a panel of 26 monoclonal antibodies (MAbs) against the VP1 capsid protein of feline calicivirus (FCV). Two close but distinct linear epitopes were identified at the capsid outermost surface (P2 subdomain) of VP1, within the E5'HVR antigenic hypervariable region: one spanning amino acids 431-435 (PAGDY), highly conserved and recognized by non-neutralizing MAbs; and a second epitope spanning amino acids 445-451 (ITTANQY), highly variable and recognized by neutralizing MAbs. These antibodies might be valuable for diagnostic applications, as well as for further research in different aspects of the biology of FCV.

A DNA Vaccine Delivery Platform Based on Elastin-Like Recombinamer Nanosystems for Rift Valley Fever Virus.

This work analyzes the immunogenicity of six genetically engineered constructs based on elastin-like recombinamers (ELRs) fused to the Gn glycoprotein from Rift Valley fever virus (RVFV). Upon transfection, all constructs showed no effect on cell viability. While fusion constructs including ELR blocks containing hydrophobic amino acids (alanine or isoleucine) did not increase the expression of viral Gn in eukaryotic cells, glutamic acid- or valine-rich fusion proteins showed enhanced expression levels compared with the constructs encoding the viral antigen alone. However, in vivo DNA plasmid immunization assays determined that the more hydrophobic constructs reduced viremia levels after RVFV challenge to a higher extent than glutamic- or valine-rich encoding plasmids and were better inducers of cellular immunity as judged by in vitro restimulation experiments. Although the Gn-ELR fusion constructs did not surpass the protective efficacy of a plasmid vaccine expressing nonfused Gn, our results warrant further experiments directed to take advantage of the immunomodulatory potential of ELR biomaterials for improving vaccines against infectious diseases.

MVA Vectored Vaccines Encoding Rift Valley Fever Virus Glycoproteins Protect Mice against Lethal Challenge in the Absence of Neutralizing Antibody Responses.

In vitro neutralizing antibodies have been often correlated with protection against Rift Valley fever virus (RVFV) infection. We have reported previously that a single inoculation of sucrose-purified modified vaccinia Ankara (MVA) encoding RVFV glycoproteins (rMVAGnGc) was sufficient to induce a protective immune response in mice after a lethal RVFV challenge. Protection was related to the presence of glycoprotein specific CD8+ cells, with a low-level detection of in vitro neutralizing antibodies. In this work we extended those observations aimed to explore the role of humoral responses after MVA vaccination and to study the contribution of each glycoprotein antigen to the protective efficacy. Thus, we tested the efficacy and immune responses in BALB/c mice of recombinant MVA viruses expressing either glycoprotein Gn (rMVAGn) or Gc (rMVAGc). In the absence of serum neutralizing antibodies, our data strongly suggest that protection of vaccinated mice upon the RVFV challenge can be achieved by the activation of cellular responses mainly directed against Gc epitopes. The involvement of cellular immunity was stressed by the fact that protection of mice was strain dependent. Furthermore, our data suggest that the rMVA based single dose vaccination elicits suboptimal humoral immune responses against Gn antigen since disease in mice was exacerbated upon virus challenge in the presence of rMVAGnGc or rMVAGn immune serum. Thus, Gc-specific cellular immunity could be an important component in the protection after the challenge observed in BALB/c mice, contributing to the elimination of infected cells reducing morbidity and mortality and counteracting the deleterious effect of a subneutralizing antibody immune response.

A bivalent B-cell epitope dendrimer peptide can confer long-lasting immunity in swine against foot-and-mouth disease.

Foot-and-mouth disease virus (FMDV) causes a widely extended contagious disease of livestock. We have previously reported that a synthetic dendrimeric peptide, termed B2 T(mal), consisting of two copies of a B-cell epitope [VP1(140-158)] linked through maleimide groups to a T-cell epitope [3A(21-35)] of FMDV, elicits potent B- and T-cell-specific responses and confers solid protection in pigs to type O FMDV challenge. Longer duration of the protective response and the possibility of inducing protection after a single dose are important requirements for an efficient FMD vaccine. Herein, we show that administration of two doses of B2 T(mal) elicited high levels of specific total IgGs and neutralizing antibodies that lasted 4-5 months after the peptide boost. Additionally, concomitant levels of IFN-γ-producing specific T cells were observed. Immunization with two doses of B2 T(mal) conferred a long-lasting reduced susceptibility to FMDV infection, up to 136 days (19/20 weeks) post-boost. Remarkably, a similar duration of the protective response was achieved by a single dose of B2 T(mal). The effect on the B2 T(mal) vaccine of RNA transcripts derived from non-coding regions in the FMDV genome, known to enhance the immune response and protection induced by a conventional inactivated vaccine, was also analysed. The contribution of our results to the development of FMD dendrimeric vaccines is discussed.

A Hyper-Attenuated Variant of Rift Valley Fever Virus Generated by a Mutagenic Drug (Favipiravir) Unveils Potential Virulence Markers.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes Rift Valley fever (RVF), a zoonotic disease of wild and domestic ruminants, causing serious economic losses and a threat to human health that could be controlled by vaccination. Though RVF vaccines are available for livestock, no RVF vaccines have been licensed for veterinary use in non-endemic countries nor for human populations in RVF risk areas. In a recent work, we showed that favipiravir, a promising drug with antiviral activity against a number of RNA viruses, led to the extinction of RVFV from infected cell cultures. Nevertheless, certain drug concentrations allowed the recovery of a virus variant showing increased resistance to favipiravir. In this work, we characterized this novel resistant variant both at genomic and phenotypic level in vitro and in vivo. Interestingly, the resistant virus displayed reduced growth rates in C6/36 insect cells but not in mammalian cell lines, and was highly attenuated but still immunogenic in vivo. Some amino acid substitutions were identified in the viral RNA-dependent RNA-polymerase (RdRp) gene and in the virus encoded type I-interferon (IFN-I) antagonist NSs gene, in catalytic core motifs and nuclear localization associated positions, respectively. These data may help to characterize novel potential virulence markers, offering additional strategies for further safety improvements of RVF live attenuated vaccine candidates.

Lethal Mutagenesis of Rift Valley Fever Virus Induced by Favipiravir.

Rift Valley fever virus (RVFV) is an emerging, mosquito-borne, zoonotic pathogen with recurrent outbreaks taking a considerable toll in human deaths in many African countries, for which no effective treatment is available. In cell culture studies and with laboratory animal models, the nucleoside analogue favipiravir (T-705) has demonstrated great potential for the treatment of several seasonal, chronic, and emerging RNA virus infections in humans, suggesting applicability to control some viral outbreaks. Treatment with favipiravir was shown to reduce the infectivity of Rift Valley fever virus both in cell cultures and in experimental animal models, but the mechanism of this protective effect is not understood. In this work, we show that favipiravir at concentrations well below the toxicity threshold estimated for cells is able to extinguish RVFV from infected cell cultures. Nucleotide sequence analysis has documented RVFV mutagenesis associated with virus extinction, with a significant increase in G to A and C to U transition frequencies and a decrease of specific infectivity, hallmarks of lethal mutagenesis.

Preserved immunogenicity of an inactivated vaccine based on foot-and-mouth disease virus particles with improved stability.

Foot-and-mouth disease virus (FMDV) is the etiological agent of a highly contagious disease that affects important livestock species. Vaccines based on inactivated FMDV virions provide a useful tool for the control of this pathogen. However, long term storage at 4°C (the temperature for vaccine storage) or ruptures of the cold chain, provoke the dissociation of virions, reducing the immunogenicity of the vaccine. An FMDV mutant carrying amino acid replacements VP1 N17D and VP2 H145Y isolated previously rendered virions with increased resistance to dissociation at 4°C. We have evaluated the immunogenicity in swine (a natural FMDV host) of a chemically inactivated vaccine based on this mutant. The presence of these amino acid substitutions did not compromise the immunological potential, including its ability to elicit neutralizing antibodies. These results support the feasibility of this kind of mutants with increased capsid stability as suitable viruses for producing improved FMDV vaccines.

Toxicity of silver nanoparticles in biological systems: Does the complexity of biological systems matter?

Currently, nanomaterials are more frequently in our daily life, specifically in biomedicine, electronics, food, textiles and catalysis just to name a few. Although nanomaterials provide many benefits, recently their toxicity profiles have begun to be explored. In this work, the toxic effects of silver nanoparticles (35nm-average diameter and Polyvinyl-Pyrrolidone-coated) on biological systems of different levels of complexity was assessed in a comprehensive and comparatively way, through a variety of viability and toxicological assays. The studied organisms included viruses, bacteria, microalgae, fungi, animal and human cells (including cancer cell lines). It was found that biological systems of different taxonomical groups are inhibited at concentrations of silver nanoparticles within the same order of magnitude. Thus, the toxicity of nanomaterials on biological/living systems, constrained by their complexity, e.g. taxonomic groups, resulted contrary to the expected. The fact that cells and virus are inhibited with a concentration of silver nanoparticles within the same order of magnitude could be explained considering that silver nanoparticles affects very primitive cellular mechanisms by interacting with fundamental structures for cells and virus alike.

Combined administration of synthetic RNA and a conventional vaccine improves immune responses and protection against foot-and-mouth disease virus in swine.

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease and a major concern in animal health worldwide. We have previously reported the use of RNA transcripts mimicking structural domains in the non-coding regions of the FMDV RNA as potent type-I interferon (IFN) inducers showing antiviral effect in vivo, as well as their immunomodulatory properties in combination with an FMD vaccine in mice. Here, we describe the enhancing effect of RNA delivery on the immunogenicity and protection induced by a suboptimal dose of a conventional FMD vaccine in pigs. Animals receiving the RNA developed earlier and higher levels of neutralizing antibodies against homologous and heterologous isolates, compared to those immunized with the vaccine alone, and had higher anti-FMDV titers at late times post-vaccination. RNA delivery also induced higher specific T-cell response and protection levels against FMDV challenge. Peripheral blood mononuclear cells from pigs inoculated with RNA and the vaccine had a higher IFN-γ specific response than those from pigs receiving the vaccine alone. When challenged with FMDV, all three animals immunized with the conventional vaccine developed antibodies to the non-structural viral proteins 3ABC and two of them developed severe signs of disease. In the group receiving the vaccine together with the RNA, two pigs were fully protected while one showed delayed and mild signs of disease. Our results support the immunomodulatory effect of these RNA molecules in natural hosts and suggest their potential use for improvement of FMD vaccines strategies.

Potential application of silver nanoparticles to control the infectivity of Rift Valley fever virus in vitro and in vivo.

In this work we have tested the potential antiviral activity of silver nanoparticles formulated as Argovit™ against Rift Valley fever virus (RVFV). The antiviral activity of Argovit was tested on Vero cell cultures and in type-I interferon receptor deficient mice (IFNAR (-/-) mice) by two different approaches: (i) different dilutions of Argovit were added to previously infected cells or administrated to animals infected with a lethal dose of virus; (ii) virus was pre-incubated with different dilutions of Argovit before inoculation in mice or cells. Though the ability of silver nanoparticles to control an ongoing RVFV infection in the conditions tested was limited, the incubation of virus with Argovit before the infection led to a reduction of the infectivity titers both in vitro and in vivo. These results reveal the potential application of silver nanoparticles to control the infectivity of RVFV, which is an important zoonotic pathogen.

Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation.

The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs) in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs).

Protection against Rift Valley fever virus infection in mice upon administration of interferon-inducing RNA transcripts from the FMDV genome.

In this work we have addressed the effect of synthetic, non-infectious, RNA transcripts, mimicking structural domains of the non-coding regions (NCRs) of the foot-and-mouth disease virus (FMDV) genome on the infection of mice with Rift Valley fever virus (RVFV). Groups of 5 mice were inoculated intraperitoneally (i.p.) with 200 µg of synthetic RNA resembling the 5'-terminal S region, the internal ribosome entry site (IRES) or the 3'-NCR of the FMDV genome. RNA inoculation was performed 24h before (-24 h), 24 h after (+24 h) or simultaneously to the challenge with a lethal dose of RVFV. Administration of the IRES RNA afforded higher survival rates than administration of S or 3'NCR transcripts either at -24h or +24h after challenge. In contrast, when RNA inoculation and viral challenge were performed simultaneously, all mice survived in both IRES- and 3'NCR-inoculated groups, with an 80% survival in mice receiving the S RNA. Among survivors, a complete correlation between significant anti-RVFV circulating antibody titers and resistance to a second lethal challenge with the virus was observed, supporting a limited viral replication in the RNA-inoculated animals upon the first challenge. All three RNA transcripts were able to induce the production of systemic antiviral and pro-inflammatory cytokines. These data show that triggering of intracellular pathogen sensing pathways constitutes a promising approach towards development of novel RVF preventive or therapeutic strategies.

Delivery of synthetic RNA can enhance the immunogenicity of vaccines against foot-and-mouth disease virus (FMDV) in mice.

We have recently described the antiviral effect in mice of in vitro-transcribed RNAs mimicking structural domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome RNA. These small, synthetic and non-infectious RNA molecules (ncRNAs) are potent type-I interferon (IFN) inducers in vivo. In this work, the immunomodulatory effect of the ncRNA corresponding to the internal ribosome entry site (IRES) on immunization with two different FMD vaccine formulations, both based on inactivated virus, including or not a commercial adjuvant, was analyzed in the mice model. The effect of the time interval between RNA inoculation and immunization was also studied. RNA delivery consistently increased the titers of specific anti-FMDV antibodies, including neutralizing antibodies, elicited after vaccination. Moreover, at day 2 after immunization, significant differences in mean antibody titers could be detected between the groups of mice receiving either vaccine co-administered with the RNA and the control group, unlike those immunized with the vaccine alone. When vaccinated mice were challenged with FMDV, the mean values of viral load were lower in the groups receiving the RNA together with the vaccine. Our results show the enhancing effect of the IRES RNA on the immune response elicited after vaccination and suggest the potential of this molecule as an adjuvant for new FMD vaccine design.

A single immunization with MVA expressing GnGc glycoproteins promotes epitope-specific CD8+-T cell activation and protects immune-competent mice against a lethal RVFV infection.

BACKGROUND: Rift Valley fever virus (RVFV) is a mosquito-borne pathogen causing an important disease in ruminants often transmitted to humans after epizootic outbreaks in African and Arabian countries. To help combat the spread of the disease, prophylactic measures need to be developed and/or improved. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we evaluated the immunogenicity and protective efficacy of recombinant plasmid DNA and modified vaccinia virus Ankara (rMVA) vectored vaccines against Rift Valley fever in mice. These recombinant vaccines encoded either of two components of the Rift Valley fever virus: the viral glycoproteins (Gn/Gc) or the nucleoprotein (N). Following lethal challenge with live RVFV, mice immunized with a single dose of the rMVA-Gn/Gc vaccine showed no viraemia or clinical manifestation of disease, but mounted RVFV neutralizing antibodies and glycoprotein specific CD8+ T-cell responses. Neither DNA-Gn/Gc alone nor a heterologous prime-boost immunization schedule (DNA-Gn/Gc followed by rMVAGn/Gc) was better than the single rMVA-Gn/Gc immunization schedule with regards to protective efficacy. However, the rMVA-Gn/Gc vaccine failed to protect IFNAR(-/-) mice upon lethal RVFV challenge suggesting a role for innate responses in protection against RVFV. Despite induction of high titer antibodies against the RVFV nucleoprotein, the rMVA-N vaccine, whether in homologous or heterologous prime-boost schedules with the corresponding recombinant DNA vaccine, only conferred partial protection to RVFV challenge. CONCLUSIONS/SIGNIFICANCE: Given the excellent safety profile of rMVA based vaccines in humans and animals, our data supports further development of rMVA-Gn/Gc as a vaccine strategy that can be used for the prevention of Rift Valley fever in both humans and livestock.