Pediatric COVID-19 patients in South Brazil show abundant viral mRNA and strong specific anti-viral responses.

COVID-19 manifests as a milder disease in children than adults, but the underlying mechanisms are not fully characterized. Here we assess the difference in cellular or humoral immune responses of pediatric and adult COVID-19 patients to see if these factors contribute to the severity dichotomy. Children's non-specific immune profile is dominated by naive lymphocytes and HLA-DRhighCX3CR1low dendritic cells; meanwhile, children show strong specific antibody and T cell responses for viral structural proteins, with their T cell responses differing from adults by having weaker CD8+TNF+ T cells responses to S peptide pool but stronger responses to N and M peptide pools. Finally, viral mRNA is more abundant in pediatric patients. Our data thus support a scenario in which SARS-CoV-2 infected children contribute to transmission yet are less susceptible to COVID-19 symptoms due to strong and differential responses to the virus.

Assessing the antigenicity of different VP3 regions of infectious bursal disease virus in chickens from South Brazil.

BACKGROUND: Infectious bursal disease (IBD), also known as Gumboro disease, is a viral infection that causes mortality and immunosuppression in chickens (Gallus gallus). VP2 and VP3 are the major structural viral capsid components and are the most immunogenic proteins of IBD virus (IBDV). Reliable diagnostic tests using VP2 and VP3 produced in heterologous systems are important tools to control this infection. One advantage of an IBD diagnostic based on VP3, over those that use VP2, is that VP3 has linear epitopes, enabling its production in bacteria. RESULTS: We tested the suitability of recombinant VP3 (rVP3) as a diagnostic reagent in an enzyme-linked immunosorbent assay (ELISA). Compared with a commercial test, rVP3 ELISA showed high sensitivity and specificity as a diagnostic tool for vaccinated animals. In addition, rVP3, but not the commercial ELISA, was able to detect antibodies in nonvaccinated chickens, probably developed against circulating IBDV strains. It was possible the assessment of VP3 regions antigenicity using chicken antisera. CONCLUSIONS: The full-length recombinant VP3 can be used to assess post vaccination immunological status of chickens and its production is feasible and inexpensive. The evaluation of VP3 regions as candidates for general use in the diagnosis of IBD in chickens should be conducted with caution. Our work was the first to identify several regions of VP3 recognized by chicken antibodies.

Detection of Antibodies against Hepatitis A Virus (HAV) by a Surface Plasmon Resonance (SPR) Biosensor: A New Diagnosis Tool Based on the Major HAV Capsid Protein VP1 (SPR-HAVP1).

Hepatitis A (HA) is an acute human infectious disease caused by a positive single-stranded RNA virus (HAV). It is mainly acquired through the fecal-oral route and is primarily spread by contact between people and exposure to contaminated water and food. Recently, large outbreaks of HA have been reported by low and moderate endemicity countries, emphasizing its importance in public health and the need for rapid and large-scale diagnostic tests to support public health decisions on HA. This work proposes a new tool for HAV diagnosis based on the association of surface plasmonic resonance with major capsid protein VP1 (SPR-HAVP1 assay), detecting IgM antibodies for HAV in human serum samples. Structural analyses of VP1 B-lymphocyte epitopes showed continuous and discontinuous epitopes. The discontinuous epitopes were identified in the N-terminal region of the VP1 protein. Both epitope types in the VP1 protein were shown by the reactivity of VP1 in native and denaturing conditions to IgM anti-HAV, which was favorable to tests of VP1 in the SPR assays. SPR-HAVP1 assays showed good performance in the detection of IgM polyclonal antibody anti-HAV. These assays were performed using a COOH5 sensor chip functionalized with VP1 protein. The sensorgram record showed a significant difference between positive and negative serum samples, which was confirmed by analysis of variation of initial and final dissociation values through time (ΔRUd/t). The data gathered here are unequivocal evidence that the SPR-HAVP1 strategy can be applied to detect IgM antibodies in human serum positive to the HAV. This is a new tool to be explored to diagnose human HAV infections.

Identification of promiscuous T cell epitopes on Mayaro virus structural proteins using immunoinformatics, molecular modeling, and QM:MM approaches.

The Mayaro virus (MAYV) belongs to genus Alphavirus (family Togaviridae) and has been reported in several countries, especially in tropical regions of America. Due to its outbreaks and potential lack of medication, an effective vaccine formulation is strongly required. This study aimed to predict promiscuous T cell epitopes from structural polyproteins of MAYV using an immunoinformatics approach. For this purpose, consensus sequences were used to identify short protein sequences capable of binding to MHC class I and class II alleles. Our analysis pointed out 4 MHC-I/TCD8+ and 21 MHC-II/TCD4+ epitopes on capside (1;3), E1 (2;5), E2 (1;10), E3 (0;2), and 6 K (0;1) proteins. These predicted epitopes were characterized by high antigenicity, immunogenicity, conservancy, non-allergenic, non-toxic, and good population coverage rate values for North and South American geographical areas. Afterwards, we used the crystal structure of human toll-like receptor 3 (TLR3) ectodomain as a template to predict, through docking essays, the placement of a vaccine prototype at the TLR3 receptor binding site. Finally, classical and quantum mechanics/molecular mechanics (QM:MM) computations were employed to improve the quality of docking calculations, with the QM part of the simulations being accomplished by using the density functional theory (DFT) formalism. These results provide important insights into the advancement of diagnostic platforms, the development of vaccines, and immunotherapeutic interventions.

A recombinant avian antibody against VP2 of infectious bursal disease virus protects chicken from viral infection.

A stable cell-line was established that expressed the recombinant avian antibody (rAb) against the infectious bursal disease virus (IBDV). rAb exhibited neutralization activity to IBDV-B87 strain in DF1 cells. The minimum rAb concentration required for inhibition of the cytopathic effect (CPE) was 1.563µg/mL. To test the efficacy of rAb, a 168-h cohabitation challenge experiment was performed to transmit the disease from the chickens challenged with vvIBDV (HLJ0504 strain) to three test groups of chickens, i.e. (1) chickens treated with rAb, (2) chickens treated with yolk antibody, and (3) non-treatment chickens. The survival rates of chickens treated with rAb, yolk antibody and without treatment were 73%, 67% and 20%, respectively. Another batch of chickens was challenged with IBDV (BC6/85 strain) and then injected with rAb (1.0mg/kg) 6, 24 and 36h post-challenge. Non-treatment chickens had 100% morbidity, whereas those administered with rAb exhibited only 20% morbidity. Morbidity was evaluated using clinical indicators and bursal histopathological section. This study provides a new approach to treating IBDV and the rAb represents a promising candidate for this IBDV therapy.

Recombinant vesicular stomatitis virus-based dengue-2 vaccine candidate induces humoral response and protects mice against lethal infection.

Dengue is the most important arbovirus disease throughout the world and it is responsible for more than 500,000 dengue hemorrhagic cases and 22,000 deaths every year. One vaccine was recently licensed for human use in Brazil, Mexico and Philippines and although at least seven candidates have been in clinical trials the results of the most developed CYD vaccine have demonstrated immunization problems, such as uneven protection and interference between serotypes. We constructed a vaccine candidate based on vesicular stomatitis virus (VSV) expression of pre-membrane (prM) and envelope (E) proteins of dengue-2 virus (DENV-2) and tested it in mice to evaluate immunogenicity and protection against DENV-2 infection. VSV has been successfully used as vaccine vectors for several viruses to induce strong humoral and cellular immune responses. The VSV-DENV-2 recombinant was constructed by inserting the DENV-2 structural proteins into a VSV plasmid DNA for recombinant VSV-DENV-2 recovery. Infectious recombinant VSV viruses were plaque purified and prM and E expression were confirmed by immunofluorescence and radiolabeling of proteins of infected cells. Forty Balb/C mice were inoculated through subcutaneous (s.c.) route with VSV-DENV-2 vaccine in a two doses schedule 15 d apart and 29 d after first inoculation, sera were collected and the mice were challenged with 50 lethal doses (LD50) of a neurovirulent DENV-2. The VSV-DENV-2 induced anti-DENV-2 antibodies and protected animals in the challenge experiment comparable to DENV-2 immunization control group. We conclude that VSV is a promising platform to test as a DENV vaccine and perhaps against others Flaviviridae.

Importance of foot and mouth disease vaccine purity in interpreting serological surveys.

The aim of this study was to determine whether the degree of purity achieved in conventional vaccines against the foot and mouth disease virus in Argentina interferes with the interpretation of seroepidemiological surveys for confirming the absence of viral activity, which are performed to support the recognition of free zones practising vaccination. The evaluation of 168 vaccine series due to be marketed in Argentina (2006-2012) and subjected to official control testing in cattle, as well as repeated vaccination of cattle and other species using vaccines with high antigen concentrations, demonstrated that they did not induce antibodies to non-structural proteins (NSPs). The results show clearly that vaccines with satisfactory potency do not induce a response to NSPs, even by forcing the immune response through more concentrated doses with multiple valences and revaccination protocols at shorter irtervals than in vaccination campaigns. These results confirm that the vaccines used in routine vaccination programmes have a degree of antigen purification consistent with the needs observed on the basis of sampling for serological surveillance. Moreover, serological surveys conducted in 2006-2011 by Argentina's official Veterinary Services--the National Health and Agrifood Quality Service (SENASA)--on more than 23,000 sera per year from cattle included in the vaccination programme, in order to confirm the absence of virus circulation, revealed an average 0.05% of reactive results, consistent with the specificity of the tests. In conclusion, the vaccines produced by conventional methods and with proven potencythat are available in Argentina are sufficiently purified to ensure thatthey do not interfere with the interpretation of sampling for serological surveillance performed to support the recognition of FMD-free zones practising vaccination.

Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens.

Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.

Mapping of CD8 T cell epitopes in human respiratory syncytial virus L protein.

OBJECTIVES: Since it has been reported that in humans there is a relationship between human respiratory syncytial virus (hRSV)-specific cytotoxic T lymphocytes and symptom reduction, and that the polymerase (structural L protein) is highly conserved among different strains, this work aimed to identify the CD8 T cell epitopes H-2(d) restricted within the L sequence for immunization purposes. METHODS: We screened the hRSV strain A2 L protein sequence using two independent algorithms, SYFPEITHI and PRED/(BALB/c), to predict CD8 T cell epitopes. The selected peptides were synthesized and used to immunize BALB/c mice for the evaluation of T cell response. The production of IFN-γ from splenocytes of hRSV-infected animals stimulated by these peptides was assayed by ELISPOT. RESULTS: Nine peptides showing the best binding scores to the BALB/c MHC-I molecules (H-2K(d), L(d) and D(d)) were selected. Sequence homology analysis showed that these sequences are conserved among different hRSV strains. Two of these peptides induced significant IFN-γ production by ex vivo-stimulated T cells. CONCLUSIONS: Our results indicate that the hRSV L protein contains H-2(d)-restricted epitopes.

Immunologic cross-reactivity between Muscovy duck parvovirus and goose parvovirus on the basis of epitope prediction.

Through bioinformatic prediction, between Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV), there were one epitope AA503-509 (RANEPKE) on non-structural protein and three epitopes AA426-430 (SQDLD), 540-544 (DPYRS), 685-691 (KENSKRW) on structural protein might cross-react with each other. Furthermore, the four epitops were expressed in Escherichia coli. All the four recombinant proteins could react with GPV-antisera and MDPV-antisera in Western blot.

Transient expression of VP2 in Nicotiana benthamiana and its use as a plant-based vaccine against infectious bursal disease virus.

Infectious Bursal Disease Virus (IBDV) is the etiological agent of an immunosuppressive and highly contagious disease that affects young birds. This disease causes important economic losses in the poultry industry worldwide. The VP2 protein has been used for the development of subunit vaccines in a variety of heterologous platforms. In this context, the aim of this study was to investigate VP2 expression and immunogenicity using an experimental plant-based vaccine against IBDV. We determined that the agroinfiltration of N. benthamiana leaves allowed the production of VP2 with no apparent change on its conformational epitopes. Chickens intramuscularly immunized in a dose/boost scheme with crude concentrated extracts developed a specific humoral response with viral neutralizing ability. Given these results, it seems plausible for a plant-based vaccine to have a niche in the veterinary field. Thus, plants can be an adequate system of choice to produce immunogens against IBDV.

Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens.

A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry.

Vesicular Stomatitis Virus glycoprotein G carrying a tandem dimer of Foot and Mouth Disease Virus antigenic site A can be used as DNA and peptide vaccine for cattle.

Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139-149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an "expected percentage of protection" above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle.

Single dose adenovirus vectored vaccine induces a potent and long-lasting immune response against rabbit hemorrhagic disease virus after parenteral or mucosal administration.

Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 10(8) gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 10(9)GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.

Dengue-2 structural proteins associate with human proteins to produce a coagulation and innate immune response biased interactome.

BACKGROUND: Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem. METHODS: A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology. RESULTS: Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis. CONCLUSIONS: Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti-viral response processes, and predicts that the interaction of dengue proteins with a proposed human protein interaction network produces a modified biological outcome that may be behind the hallmark pathologies of dengue infection.

A DNA vaccine candidate encoding the structural prM/E proteins elicits a strong immune response and protects mice against dengue-4 virus infection.

A DNA vaccine expressing dengue-4 virus premembrane (prM) and envelope (E) genes was produced by inserting these genes into a mammalian expression plasmid (pCI). Following a thorough screening, including confirmation of protein expression in vitro, a recombinant clone expressing these genes was selected and used to immunize BALB/c mice. After 3 immunizations all the animals produced detectable levels of neutralizing antibodies against dengue-4 virus. The cytokines levels and T cell proliferation, detected ex vivo from the spleen of the immunized mice, showed that our construction induced substantial immune stimulation after three doses. Even though the antibody levels, induced by our DNA vaccine, were lower than those obtained in mice immunized with dengue-4 virus the levels of protection were high with this vaccine. This observation is further supported by the fact that 80% of the vaccine immunized group was protected against lethal challenge. In conclusion, we developed a DNA vaccine employing the genes of the prM and E proteins from dengue-4 virus that protects mice against this virus.

Immunogenicity and protective efficacy of a DNA vaccine against Venezuelan equine encephalitis virus aerosol challenge in nonhuman primates.

A study to evaluate the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus (VEEV) DNA vaccine in an aerosol model of nonhuman primate infection was performed. Cynomolgus macaques vaccinated with a plasmid expressing the 26S structural genes of VEEV subtype IAB by particle-mediated epidermal delivery (PMED) developed virus-neutralizing antibodies. No serum viremia was detected in two out of three macaques vaccinated with the VEEV DNA after aerosol challenge with homologous virus, while one displayed a low viremia on a single day postchallenge. In contrast, all three macaques vaccinated with empty vector DNA developed a high viremia that persisted for at least 3 days after challenge. In addition, macaques vaccinated with the VEEV DNA had reduced febrile reactions, lymphopenia, and clinical signs of disease postchallenge as compared to negative control macaques. Therefore, although the sample size was small in this pilot study, these results indicate that a VEEV DNA vaccine administered by PMED can at least partially protect nonhuman primates against an aerosol VEEV challenge.

Improved efficacy of a gene optimised adenovirus-based vaccine for venezuelan equine encephalitis virus.

BACKGROUND: Optimisation of genes has been shown to be beneficial for expression of proteins in a range of applications. Optimisation has increased protein expression levels through improved codon usage of the genes and an increase in levels of messenger RNA. We have applied this to an adenovirus (ad)-based vaccine encoding structural proteins (E3-E2-6K) of Venezuelan equine encephalitis virus (VEEV). RESULTS: Following administration of this vaccine to Balb/c mice, an approximately ten-fold increase in antibody response was elicited and increased protective efficacy compared to an ad-based vaccine containing non-optimised genes was observed after challenge. CONCLUSION: This study, in which the utility of optimising genes encoding the structural proteins of VEEV is demonstrated for the first time, informs us that including optimised genes in gene-based vaccines for VEEV is essential to obtain maximum immunogenicity and protective efficacy.

Molecular and antigenic characterization of rabbit hemorrhagic disease virus isolated in Cuba indicates a distinct antigenic subtype.

Phylogenetic analyses conducted on isolates of rabbit hemorrhagic disease virus (RHDV) from throughout the world have shown well-defined genogroups comprising representative strains of the virus and antigenic variants. In this work, we have isolated and characterized RHDV from the major epizootic that occurred in Cuba in 2004-2005. Sequence analysis of the capsid protein gene and antigenic characterization of this strain has allowed its inclusion as a member of the distinct RHDVa subtype. We also found that specific antibodies directed against RHDV reference strains bound to the Cuban isolate in a competition ELISA and inhibited virus hemagglutination in vitro. This is the second report on the molecular characterization of RHDVa circulating in the American region.