Relevance of the N-terminal and major hydrophobic domains of non-structural protein 3A in the replicative process of a DNA-launched foot-and-mouth disease virus replicon.

A foot-and-mouth disease virus (FMDV) DNA-launched reporter replicon containing a luciferase gene was used to assess the impact of non-structural (NS) protein 3A on viral replication. Independent deletions within the N-terminal region (amino acid [aa] residues 6 to 24) and the central hydrophobic region (HR, aa 59 to 76) of FMDV NS protein 3A were engineered, and luciferase activity in lysates of control and mutated replicon-transfected cells was measured. Triple alanine replacements of the N-terminal triplet Arg 18- His 19 -Glu 20 and a single alanine substitution of the highly charged Glu 20 residue both resulted in a 70-80% reduction in luciferase activity when compared with wild-type controls. Alanine substitution of the 17 aa present in the central HR, on the other hand, resulted in complete inhibition of luciferase activity and in the accumulation of the mutated 3A within the cell nucleus according to immunofluorescence analysis. Our results suggest that both the aa sequence around the putatively exposed hydrophilic E20 residue at the N-terminus of the protein and the hydrophobic tract located between aa 59 and 76 are of major relevance for maintaining the functionality of the 3A protein and preventing its mislocalization into the cell nucleus.

Recombinant foot-and-mouth disease virus (FMDV) non-structural protein 3A fused to enhanced green fluorescent protein (EGFP) as a candidate probe to identify FMDV-infected cattle in serosurveys.

Recombinant protein 3A-EGFP, a fusion construct between foot-and-mouth disease virus (FMDV) non-structural protein 3A and the enhanced green fluorescent protein (EGFP) was expressed in BL21-DE3 cells. The identity of the partially purified protein 3A-EGFP was confirmed by its reactivity with sera from cattle infected with FMDV and with a monoclonal antibody specific for FMDV-3ABC (MAb3H7) in Western blot assays. No reactivity was observed with sera from uninfected vaccinated animals. The performance of 3A-EGFP as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) was assessed and compared with that of a previously developed and validated capture ELISA that uses a 3ABC recombinant antigen (3ABC ELISA) and has been widely applied for serological surveys in Argentina. Parallel analysis of strongly and weakly positive reference sera from infected animals and 329 serum samples from uninfected vaccinated cattle showed that the 3A-EGFP antigen unequivocally identifies sera from FMDV-infected cattle with similar performance to its 3ABC counterpart. The 3A-EGFP ELISA is simpler and faster to perform than the 3ABC ELISA, since it does not require a capture step with a specific antibody. Moreover, the expression and storage of the recombinant 3A-EGFP is simplified by the absence of residual autoproteolytic activity associated to the 3C sequence. We conclude that the 3A-EGFP ELISA constitutes a promising screening method in serosurveys to determine whether or not animals are infected with FMDV.

Genetic characterization of South American infectious bursal disease virus reveals the existence of a distinct worldwide-spread genetic lineage.

Infectious bursal disease virus (IBDV) is one of the most concerning health problems for world poultry production. IBDVs comprise four well-defined evolutionary lineages known as classic (c), classic attenuated (ca), variant (va) and very virulent (vv) strains. Here, we characterized IBDVs from South America by the genetic analysis of both segments of the viral genome. Viruses belonging to c, ca and vv strains were unambiguously classified by the presence of molecular markers and phylogenetic analysis of the hypervariable region of the vp2 gene. Notably, the majority of the characterized viruses (9 out of 15) could not be accurately assigned to any of the previously described strains and were then denoted as distinct (d) IBDVs. These dIBDVs constitute an independent evolutionary lineage that also comprises field IBDVs from America, Europe and Asia. The hypervariable VP2 sequence of dIBDVs has a unique and conserved molecular signature (272T, 289P, 290I and 296F) that is a diagnostic character for classification. A discriminant analysis of principal components (DAPC) also identified the dIBDVs as a cluster of genetically related viruses separated from the typical strains. DAPC and genetic distance estimation indicated that the dIBDVs are one of the most genetically divergent IBDV lineages. The vp1 gene of the dIBDVs has non-vvIBDV markers and unique nucleotide and amino acid features that support their divergence in both genomic segments. The present study suggests that the dIBDVs comprise a neglected, highly divergent lineage that has been circulating in world poultry production since the early time of IBDV emergence.

HSV-1 amplicon vectors launch the production of heterologous rotavirus-like particles and induce rotavirus-specific immune responses in mice.

Virus-like particles (VLPs) are promising vaccine candidates because they represent viral antigens in the authentic conformation of the virion and are therefore readily recognized by the immune system. As VLPs do not contain genetic material they are safer than attenuated virus vaccines. In this study, herpes simplex virus type 1 (HSV-1) amplicon vectors were constructed to coexpress the rotavirus (RV) structural genes VP2, VP6, and VP7 and were used as platforms to launch the production of RV-like particles (RVLPs) in vector-infected mammalian cells. Despite the observed splicing of VP6 RNA, full-length VP6 protein and RVLPs were efficiently produced. Intramuscular injection of mice with the amplicon vectors as a two-dose regimen without adjuvants resulted in RV-specific humoral immune responses and, most importantly, immunized mice were partially protected at the mucosal level from challenge with live wild-type (wt) RV. This work provides proof of principle for the application of HSV-1 amplicon vectors that mediate the efficient production of heterologous VLPs as genetic vaccines.

Enhancement of Th1 immune responses to recombinant influenza nucleoprotein by Ribi adjuvant.

A broad coverage influenza vaccine against multiple viral strains based on the viral nucleoprotein (NP) is a goal pursued by many laboratories. If the goal is to formulate the vaccine with recombinant NP it is essential to count on adjuvants capable of inducing cellular immunity. This work have studied the effect of the monophosphoryl lipid A and trehalose dimycolate, known as the Ribi Adjuvant System (RAS), in the immune response induced in mice immunized with recombinant NP. The NP was formulated with RAS and used to immunize BALB/c mice. Immunizations with NP-RAS increased the humoral and cellular immune responses compared to unadjuvanted NP. The predominant antibody isotype was IgG2a, suggesting the development of a Th1 response. Analysis of the cytokines from mice immunized with NP-RAS showed a significant increase in the production of IFN-g and a decreased production of IL-10 and IL-4 compared to controls without RAS. These results are similar to those usually obtained using Freund's adjuvant, known to induce Th1 and CTL responses when co-administered with purified proteins, and suggest that a similar approach may be possible to enhance the performance of a T-cell vaccine containing NP.

Study of canine parvovirus evolution: comparative analysis of full-length VP2 gene sequences from Argentina and international field strains.

The continuous emergence of new strains of canine parvovirus (CPV), poorly protected by current vaccination, is a concern among breeders, veterinarians, and dog owners around the world. Therefore, the understanding of the genetic variation in emerging CPV strains is crucial for the design of disease control strategies, including vaccines. In this paper, we obtained the sequences of the full-length gene encoding for the main capsid protein (VP2) of 11 canine parvovirus type 2 (CPV-2) Argentine representative field strains, selected from a total of 75 positive samples studied in our laboratory in the last 9 years. A comparative sequence analysis was performed on 9 CPV-2c, one CPV-2a, and one CPV-2b Argentine strains with respect to international strains reported in the GenBank database. In agreement with previous reports, a high degree of identity was found among CPV-2c Argentine strains (99.6-100% and 99.7-100% at nucleotide and amino acid levels, respectively). However, the appearance of a new substitution in the 440 position (T440A) in four CPV-2c Argentine strains obtained after the year 2009 gives support to the variability observed for this position located within the VP2, three-fold spike. This is the first report on the genetic characterization of the full-length VP2 gene of emerging CPV strains in South America and shows that all the Argentine CPV-2c isolates cluster together with European and North American CPV-2c strains.

Functional and in silico Characterization of Neutralizing Interactions Between Antibodies and the Foot-and-Mouth Disease Virus Immunodominant Antigenic Site.

Molecular knowledge of virus-antibody interactions is essential for the development of better vaccines and for a timely assessment of the spread and severity of epidemics. For foot-and-mouth disease virus (FMDV) research, in particular, computational methods for antigen-antibody (Ag-Ab) interaction, and cross-antigenicity characterization and prediction are critical to design engineered vaccines with robust, long-lasting, and wider response against different strains. We integrated existing structural modeling and prediction algorithms to study the surface properties of FMDV Ags and Abs and their interaction. First, we explored four modeling and two Ag-Ab docking methods and implemented a computational pipeline based on a reference Ag-Ab structure for FMDV of serotype C, to be used as a source protocol for the study of unknown interaction pairs of Ag-Ab. Next, we obtained the variable region sequence of two monoclonal IgM and IgG antibodies that recognize and neutralize antigenic site A (AgSA) epitopes from South America serotype A FMDV and developed two peptide ELISAs for their fine epitope mapping. Then, we applied the previous Ag-Ab molecular structure modeling and docking protocol further scored by functional peptide ELISA data. This work highlights a possible different behavior in the immune response of IgG and IgM Ab isotypes. The present method yielded reliable Ab models with differential paratopes and Ag interaction topologies in concordance with their isotype classes. Moreover, it demonstrates the applicability of computational prediction techniques to the interaction phenomena between the FMDV immunodominant AgSA and Abs, and points out their potential utility as a metric for virus-related, massive Ab repertoire analysis or as a starting point for recombinant vaccine design.

Optimized Adenoviral Vector That Enhances the Assembly of FMDV O1 Virus-Like Particles in situ Increases Its Potential as Vaccine for Serotype O Viruses.

Although replication-defective human adenovirus type 5 (Ad5) vectors that express in situ the capsid-encoding region of foot-and-mouth disease virus (FMDV) have been proven to be effective as vaccines in relevant species for several viral strains, the same result was not consistently achieved for the O1/Campos/Brazil/58 strain. In the present study, an optimization of the Ad5 system was explored and was proven to enhance the expression of FMDV capsid proteins and their association into virus-like particles (VLPs). Particularly, we engineered a novel Ad5 vector (Ad5[PVP2]OP) which harbors the foreign transcription unit in a leftward orientation relative to the Ad5 genome, and drives the expression of the FMDV sequences from an optimized cytomegalovirus (CMV) enhancer-promoter as well. The Ad5[PVP2]OP vaccine candidate also contains the amino acid substitutions S93F/Y98F in the VP2 protein coding sequence, predicted to stabilize FMD virus particles. Cells infected with the optimized vector showed an ∼14-fold increase in protein expression as compared to cells infected with an unmodified Ad5 vector tested in previous works. Furthermore, amino acid substitutions in VP2 protein allowed the assembly of FMDV O1/Campos/Brazil/58 VLPs. Evaluation of several serological parameters in inoculated mice with the optimized Ad5[PVP2]OP candidate revealed an enhanced vaccine performance, characterized by significant higher titers of neutralizing antibodies, as compared to our previous unmodified Ad5 vector. Moreover, 94% of the mice vaccinated with the Ad5[PVP2]OP candidate were protected from homologous challenge. These results indicate that both the optimized protein expression and the stabilization of the in situ generated VLPs improved the performance of Ad5-vectored vaccines against the FMDV O1/Campos/Brazil/58 strain and open optimistic expectations to be tested in target animals.

FMD vaccine matching: Inter laboratory study for improved understanding of r1 values.

Foot-and-mouth disease virus (FMDV) is a highly variable RNA virus existing as seven different serotypes. The antigenic variability between and within serotypes can limit the cross-reactivity and therefore the in vivo cross-protection of vaccines. Selection of appropriate vaccine strains is crucial in the control of FMD. Determination of indirect relationships (r1-value) between potential vaccine strains and field strains based on antibody responses against both are routinely used for vaccine matching purposes. Aiming at the investigation of the repeatability, reproducibility and comparability of r1-value determination within and between laboratories and serological tests, a small scale vaccine matching ring test for FMDV serotype A was organized. Well-characterized serum pools from cattle vaccinated with a monovalent A24/Cruzeiro/Brazil/55 (A24) FMD vaccine with known in vivo protection status (homologous and heterologous) were distributed to four laboratories to determine r1-values for the heterologous FMD strains A81/Argentina/87, A/Argentina/2000 and A/Argentina/2001 using the virus neutralization tests (VNT) and liquid phase blocking ELISA (LPBE). Within laboratories, the repeatability of r1-value determination was high for both antibody assays. VNT resulted in reproducible and comparable r1-values between laboratories, indicative of a lack of antigenic relatedness between the A24 strain and the heterologous strains tested in this work, thus corresponding to some of the in vivo findings with these strains. Using LPBE, similar trends in r1-values were observed in all laboratories, but the overall reproducibility was lower than with VNT. Inconsistencies between laboratories may at least in part be attributed to differences in LPBE protocols as well as the in preexisting information generated in each laboratory (such as antibody titer-protection correlation curves). To gain more insight in the LPBE-derived r1-values standard bovine control sera were included in the antibody assays performed in each laboratory and a standardization exercise was performed.

Rapid methodology for antigenic profiling of FMDV field strains and for the control of identity, purity and viral integrity in commercial virus vaccines using monoclonal antibodies.

Monoclonal antibodies (MAbs) developed against different foot-and-mouth disease virus (FMDV) vaccine strains were extensively used to study any possible antigenic variations during vaccine production in Argentine facilities. Additionally, a typing ELISA using strain specific MAbs was developed to detect potential cross contaminations among FMDV strains in master and working seeds with high specificity and sensitivity and to confirm strains identity in formulated vaccines. This assay was carried out for the South American strains currently in use in production facilities in Argentina (A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial) and for the strain O/Taiwan, produced only for export to Asia. These non-cross reactive MAbs were also used to analyze the integrity of viral particles belonging to each one of the individual strains, following isolation of 140S virions by means of sucrose density gradients from the aqueous phase of commercial polyvalent vaccines. Antigenic profiles were defined for FMDV reference strains using panels of MAbs, and a coefficient of correlation of reactivity with these panels was calculated to establish consistent identity upon serial passages of master and production seeds. A comparison of vaccine and field strain antigenic profiles performed using coefficients of correlation allowed the rapid identification of two main groups of serotype A viruses collected during the last FMD epidemic in Argentina, whose reactivity matched closely to A/Argentina/2000 and A/Argentina/2001 strains.

Genetic and subunit vaccines based on the stem domain of the equine influenza hemagglutinin provide homosubtypic protection against heterologous strains.

H3N8 influenza virus strains have been associated with infectious disease in equine populations throughout the world. Although current vaccines for equine influenza stimulate a protective humoral immune response against the surface glycoproteins, disease in vaccinated horses has been frequently reported, probably due to poor induction of cross-reactive antibodies against non-matching strains. This work describes the performance of a recombinant protein vaccine expressed in prokaryotic cells (ΔHAp) and of a genetic vaccine (ΔHAe), both based on the conserved stem region of influenza hemagglutinin (HA) derived from A/equine/Argentina/1/93 (H3N8) virus. Sera from mice inoculated with these immunogens in different combinations and regimes presented reactivity in vitro against highly divergent influenza virus strains belonging to phylogenetic groups 1 and 2 (H1 and H3 subtypes, respectively), and conferred robust protection against a lethal challenge with both the homologous equine strain (100%) and the homosubtypic human strain A/Victoria/3/75 (H3N2) (70-100%). Animals vaccinated with the same antigens but challenged with the human strain A/PR/8/34 (H1N1), belonging to the phylogenetic group 1, were not protected (0-33%). Combination of protein and DNA immunogens showed higher reactivity to non-homologous strains than protein alone, although all vaccines were permissive for lung infection.

Evaluation of the immunogenicity of a recombinant HSV-1 vector expressing human group C rotavirus VP6 protein.

Group C Rotavirus (RVC) has been associated globally with sporadic outbreaks of gastroenteritis in children and adults. RVC also infects animals, and interspecies transmission has been reported as well as its zoonotic potential. Considering its genetic diversity and the absence of effective vaccines, it is important and necessary to develop new generation vaccines against RVC for both humans and animals. The aim of the present study was to develop and characterize an HSV-1-based amplicon vector expressing a human RVC-VP6 protein and evaluate the humoral immune response induced after immunizing BALB/c mice. Local fecal samples positive for RVC were used for isolation and sequencing of the vp6 gene, which phylogenetically belongs to the I2 genotype. We show here that cells infected with the HSV[VP6C] amplicon vector efficiently express the VP6 protein, and induced specific anti-RVC antibodies in mice immunized with HSV[VP6C], in a prime-boost schedule. This work highlights that amplicon vectors are an attractive platform for the generation of safe genetic immunogens against RVC, without the addition of external adjuvants.

Detection by RT-PCR and genetic characterization of canine distemper virus from vaccinated and non-vaccinated dogs in Argentina.

RT-PCR was used to detect canine distemper virus (CDV) RNA in clotted blood from Argentine domestic dogs. The NP gene was detected in 73 out of 99 blood samples analyzed. The deduced amino acid sequence of these gene fragments showed 100% identity with the sequence of other wild-type and vaccine strains. A fragment of the hemagglutinin gene was amplified from 24 (32.9%) of the NP-RNA-positive clinical specimens. These H fragments were further analyzed by restriction fragment length polymorphism (RFLP) and sequencing. A single NdeI site was detected in all 24 wild-type strains but was absent in the vaccine strains. Phylogenetic analysis of the partial hemagglutinin amino acid sequences showed close clustering for local strains, clearly distinct from vaccine strains and other wild-type foreign CDV strains. One of the local strains, Arg 23, branched out of the root of the Argentine clade, close to the European strains, suggesting that two different pathogenic CDV genotypes are currently circulating in Argentina, one of them clearly predominant.

Characterization of infectious bursal disease viruses from Argentina.

Infectious bursal disease (IBD) viruses detected in commercial flocks of different regions of Argentina were analyzed by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RFLP) of a VP2 gene fragment, followed by sequence analysis. Two out of eight IBD viruses presented an SspI restriction site, typical of the very virulent phenotype. Three IBD viruses presented a SacI restriction site, typical of classic virulent strains, and one isolate presented restriction sites for both enzymes. The Argentine IBD viruses showed three different molecular patterns by RFLP with the restriction endonuclease BstNI and five different patterns with MboI. By comparison of nucleotide and deduced amino acid sequences of the hypervariable region of the VP2 protein, four Argentine viruses were found to be closely related to Brazilian subclinical strains and two isolates were found to be related to vaccine IBDV strains in use in Argentina. Strain LD9569 was genetically characterized as a very virulent strain and was found to be closely related to international and regional vvIBDV strains. This is the first report on variability of IBDV strains circulating in Argentina.

RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks.

Resurgence of canine parvovirus 2a strain in the domestic dog population from Argentina.

Ninety-three rectal swab samples were taken, from dogs suspected of canine parvovirus (CPV) infection and analyzed by PCR. A fragment of the VP2 gene, was amplified in 41 (44%) of them, resulting CPV positive samples. Sequencing analysis of these PCR products showed that 37 samples (90.2%) belonged to the CPV2c type, whereas four samples (9.8%) were identified as CPV2a, which has not been found since 2008. It was also found that 24 out of 37 CPV2c samples (65%), carried the mutation Thr440Ala, whereas this mutation was absent in the four CPV2a strains reported herein. Using phylogenetic analysis of the full length VP2 gene, which was amplified by PCR in six local samples, it was seen that CPV2a Argentine strains reported in this study, were genetically closer to a previous local CPV2a isolate (year 2003) and to a South African CPV2a strain, than to any of the recently reported Uruguayan CPV2a strains. The results obtained in this work, together with those reported previously in Uruguay strongly suggest that, in spite of the geographical proximity, wild type CPV strains undergo different evolutive pathways in each country, resulting in the prevalence of different strains in related dog populations. Further extensive epidemiological studies are needed in order to improve the understanding of CPV evolution.

Evaluation of the immune response to Anaplasma marginale MSP5 protein using a HSV-1 amplicon vector system or recombinant protein.

Anaplasma marginale is an intraerythrocytic vector-borne infectious agent of cattle. Immunization with the current vaccine, based on parasitized erythrocytes with live Anaplasma centrale, shows some constraints and confers partial protection, suggesting the feasibility for the development of new generation of vaccines. The aim of the present study was to assess the effect of sequential immunization of BALB/c mice, with herpesvirus amplicon vector-based vaccines combined with protein-based vaccines, on the quality of the immune response against the major surface protein 5 of A. marginale. The highest antibody titers against MSP5 were elicited in mice that received two doses of adjuvanted recombinant protein (p < 0.0001). Mice treated with a heterologous prime-boost strategy generated sustained antibody titers at least up to 200 days, and a higher specific cellular response. The results presented here showed that sequential immunization with HSV-based vectors and purified antigen enhances the quality of the immune response against A. marginale.

Emergence of antigenic variants of Foot-and-Mouth Disease Virus serotype O in Ecuador and preliminary evaluation of a field strain as a vaccine candidate.

Foot-and-Mouth Disease Virus serotype O has been circulating regularly throughout most provinces of Ecuador, one of the two South American countries that still remain endemic, although satisfactory vaccination coverage was reported. This study concentrates in the characterization of isolates collected during 2008-2011, focusing particularly on the antigenic and immunogenic relationships of the field viruses with the O1/Campos vaccine strain in use in the region and with an experimental vaccine formulated with a representative strain of the 2010 epidemic. The results established that antigenically divergent variants poorly protected by the vaccine in use emerged and co-circulated in a limited period of time. A monovalent vaccine formulated with the representative 2010 strain elicited high antibody titers and protected against challenge with homologous virus. In addition, cross-reactive antibodies to predominant viruses in the region were established. In overall this study indicates the ability of the virus to diversify under field conditions in which a vaccine strain with poor match is applied, and the potential of the selected 2010 field virus as a vaccine candidate for incorporation into strategic antigen banks and/or for addition to current formulations for systematic vaccination, in order to prevent the emergence of even more divergent isolates in the future.

Tandem repeats of the extracellular domain of Matrix 2 influenza protein exposed in Brucella lumazine synthase decameric carrier molecule induce protection in mice.

The antigenic variation of influenza virus represents a major prevention problem. However, the ectodomain of the protein Matrix 2 (M2e) is nearly invariant in all human influenza A strains and has been considered as a promising candidate for a broadly protective vaccine because antibodies to M2e are protective in animal models. In this work we evaluated the possible use of Brucella abortus lumazine synthase protein (BLS), a highly immunogenic decameric protein, as a carrier of the M2e peptide. Chimeric proteins generated by the fusion of one or four in tandem copies of M2e to BLS were efficiently expressed in Escherichia coli and assembled in decameric subunits similarly to the wild type BLS enzyme, as demonstrated by the comparative circular dichroism spectra and size exclusion chromatography and static light scattering analysis. The M2e peptides were stably exposed at the ten N-terminal ends of each BLS molecule. Immunization of mice with purified chimeras carrying only one M2e (BLS-M2e) copy elicited a significant humoral immune response with the addition of different adjuvants. The fusion of four in tandem copies of the M2e peptide (BLS-4M2e) resulted in similar levels of humoral immune response but in the absence of adjuvant. Survival of mice challenged with live influenza virus was 100% after vaccination with BLS-4M2e adjuvanted with Iscomatrix(®) (P<0.001) and 80% when adjuvanted with alum (P<0.01), while the chimera alone protected 60% of the animals (P<0.05). The approach described in this study is intended as a contribution to the generation of universal influenza immunogens, through a simple production and purification process and using safe carriers that might eventually avoid the use of strong adjuvants.