The Unique Glycosylation at Position 986 on the E2 Glycoprotein of Classical Swine Fever Virus Is Responsible for Viral Attenuation and Protection against Lethal Challenge.

Classical swine fever (CSF) is an economically important disease of pigs caused by classical swine fever virus (CSFV). The live attenuated vaccine C-strain (also called HCLV strain) against CSF was produced by multiple passages of a highly virulent strain in rabbits. However, the molecular determinants for its attenuation and protection remain unclear. In this study, we identified a unique glycosylation at position 986 (986NYT988) on the E2 glycoprotein Domain IV of C-strain but not (986NYA988) the highly virulent CSFV Shimen strain. We evaluated the infectivity, virulence, and protective efficacy of the C-strain-based mutant rHCLV-T988A lacking the glycosylation and Shimen strain mutant rShimen-A988T acquiring an additional glycosylation at position 986. rShimen-A988T showed a significantly decreased viral replication ability in SK6 cells, while rHCLV-T988A exhibited a growth kinetics indistinguishable from that of C-strain. Removal of the C-strain glycosylation site does not affect viral replication in rabbits and the attenuated phenotype in pigs. However, rShimen-A988T was attenuated and protected the pigs from a lethal challenge at 14 days postinoculation. In contrast, the rHCLV-T988A-inoculated pigs showed transient fever, a few clinical signs, and pathological changes in the spleens upon challenge with the Shimen strain. Mechanistic investigations revealed that the unique glycosylation at position 986 influences viral spreading, alters the formation of E2 homodimers, and leads to increased production of neutralizing antibodies. Collectively, our data for the first time demonstrate that the unique glycosylation at position 986 on the E2 glycoprotein is responsible for viral attenuation and protection. IMPORTANCE Viral glycoproteins involve in infectivity, virulence, and host immune responses. Deglycosylation on the Erns, E1, or E2 glycoprotein of highly virulent classical swine fever virus (CSFV) attenuated viral virulence in pigs, indicating that the glycosylation contributes to the pathogenicity of the highly virulent strain. However, the effects of the glycosylation on the C-strain E2 glycoprotein on viral infectivity in cells, viral attenuation, and protection in pigs have not been elucidated. This study demonstrates the unique glycosylation at position 986 on the C-strain E2 glycoprotein. C-strain mutant removing the glycosylation at the site provides only partial protection against CSFV challenge. Remarkably, the addition of the glycan to E2 of the highly virulent Shimen strain attenuates the viral virulence and confers complete protection against the lethal challenge in pigs. Our findings provide a new insight into the contribution of the glycosylation to the virus attenuation and protection.

The envelope protein of tick-borne encephalitis virus influences neuron entry, pathogenicity, and vaccine protection.

BACKGROUND: Tick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection range from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known; however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection. METHOD: Mice were infected with different TBEV strains, and high virulent and low virulent TBEV strains were chosen. Sequence alignment identified differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes, mouse embryonic fibroblasts, and in vivo. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated. RESULTS: We identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild-type (WT) Torö and this correlated with enhanced pathogenicity and higher levels of viral RNA in vivo. The E protein is also the major target of neutralizing antibodies; thus, genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we chose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains. CONCLUSIONS: Our findings show that two amino acid substitutions of the E protein found in 93/783, A83T, and A463S enhanced Torö infection of neurons as well as pathogenesis and viral replication in vivo; furthermore, we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.

Genome based evolutionary lineage of SARS-CoV-2 towards the development of novel chimeric vaccine.

The present study aimed to predict a novel chimeric vaccine by simultaneously targeting four major structural proteins via the establishment of ancestral relationship among different strains of coronaviruses. Conserved regions from the homologous protein sets of spike glycoprotein, membrane protein, envelope protein and nucleocapsid protein were identified through multiple sequence alignment. The phylogeny analyses of whole genome stated that four proteins reflected the close ancestral relation of SARS-CoV-2 to SARS-COV-1 and bat coronavirus. Numerous immunogenic epitopes (both T cell and B cell) were generated from the common fragments which were further ranked on the basis of antigenicity, transmembrane topology, conservancy level, toxicity and allergenicity pattern and population coverage analysis. Top putative epitopes were combined with appropriate adjuvants and linkers to construct a novel multiepitope subunit vaccine against COVID-19. The designed constructs were characterized based on physicochemical properties, allergenicity, antigenicity and solubility which revealed the superiority of construct V3 in terms safety and efficacy. Essential molecular dynamics and normal mode analysis confirmed minimal deformability of the refined model at molecular level. In addition, disulfide engineering was investigated to accelerate the stability of the protein. Molecular docking study ensured high binding affinity between construct V3 and HLA cells, as well as with different host receptors. Microbial expression and translational efficacy of the constructs were checked using pET28a(+) vector of E. coli strain K12. However, the in vivo and in vitro validation of suggested vaccine molecule might be ensured with wet lab trials using model animals for the implementation of the presented data.

A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate.

Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.

Bioprocess optimization for purification of chimeric VLP displaying BVDV E2 antigens produced in yeast Hansenula polymorpha.

Chimeric virus-like particles (VLP) are known as promising tools in the development of safe and effective subunit vaccines. Recently, a technology platform to produce VLP based on the small surface protein (dS) of the duck hepatitis B virus was established. In this study, chimeric VLP were investigated displaying the 195 N-terminal amino acids derived from the glycoprotein E2 of the bovine viral diarrhea virus (BVDV) on their surface. Isolation of the VLP from methylotrophic yeast Hansenula polymorpha was allowed upon co-expression of wild-type dS and a fusion protein composed of the BVDV-derived antigen N-terminally fused to the dS. It was shown the VLP could be purified by a process adapted from the production of a recombinant hepatitis B VLP vaccine. However, the process essentially depended on costly ultracentrifugation which is critical for low cost production. In novel process variants, this step was avoided after modification of the initial batch capture step, the introduction of a precipitation step and adjusting the ion exchange chromatography. The product yield could be improved by almost factor 8 to 93 ± 12 mg VLP protein per 100 g dry cell weight while keeping similar product purity and antigenicity. This allows scalable and cost efficient VLP production.

Optimized Expression of Duck Tembusu Virus E Gene Delivered by a Vectored Duck Enteritis Virus In Vitro.

In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken's codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck's codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed. Then the expression cassettes pCMV-EG-polyABGH amplified from pEP-BGH-EG by PCR were inserted into US7/US8 gene intergenic region of pDEV-EF1 by two-step Red/ET recombination, 7 strain recombinant mutated BAC clones pDEV-EG carrying different E genes were constructed. Next, the recombinant viruses rDEV-EG were reconstituted from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Western blot analysis showed that E or E451 protein is expressed in rDEV-E-ori, rDEV-E-ch, rDEV-Etpa-ori, rDEV-E451-ori, rDEV-E451-dk, and rDEV-E451-ch-infected CEFs, and protein expression level in rDEV-E451-dk-infected CEFs is the highest. These studies have laid a foundation for developing bivalent vaccine controlling DEV and DTMUV infection.

Design of novel multi-epitope vaccines against severe acute respiratory syndrome validated through multistage molecular interaction and dynamics.

Severe acute respiratory syndrome (SARS) is endemic in South China and is continuing to spread worldwide since the 2003 outbreak, affecting human population of 37 countries till present. SARS is caused by the severe acute respiratory syndrome Coronavirus (SARS-CoV). In the present study, we have designed two multi-epitope vaccines (MEVs) composed of cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL) and B cell epitopes overlap, bearing the potential to elicit cellular as well as humoral immune response. We have used truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 as molecular adjuvants at N-terminal of both the MEVs. Selected overlapping epitopes of both the MEVs were further validated for stable molecular interactions with their respective human leukocyte antigen class I and II allele binders. Moreover, CTL epitopes were further studied for their molecular interaction with transporter associated with antigen processing. Furthermore, after tertiary structure modelling, both the MEVs were validated for their stable molecular interaction with Toll-like receptors 2 and 4. Codon-optimized cDNA of both the MEVs was analysed for their potential high level of expression in the mammalian cell line (Human) needed for their further in vivo testing. Overall, the present study proposes in silico validated design of two MEVs against SARS composed of specific epitopes with the potential to cause a high level of SARS-CoV specific cellular as well as humoral immune response. Communicated by Ramaswamy H. Sarma.

Construction and immunogenicity of a recombinant swinepox virus expressing a multi-epitope peptide for porcine reproductive and respiratory syndrome virus.

To characterize neutralizing mimotopes, phages were selected from a 12-mer phage display library using three anti-porcine reproductive and respiratory syndrome virus (PRRSV) neutralizing monoclonal antibodies: (1) A1; (2) A2; and (3) A7. Of these, A2 and A7 recognize the mimotope, P2, which contains the SRHDHIH motif, which has conserved consensus sequences from amino acid positions 156 to 161 in the N-terminal ectodomain of GP3. The artificial multi-epitope gene, mp2, was designed by combining three repeats of the mimotope P2. The resulting sequence was inserted into the swinepox virus (SPV) genome to construct a recombinant swinepox virus (rSPV-mp2). The rSPV-mp2 was able to stably express the multi-epitope peptide, mP2, in vitro. The rSPV-mp2 immunized pigs exhibited a significantly shorter fever duration compared with the wtSPV treated group (P < 0.05). There was an enhanced humoral and cellular immune response, decreased number of PRRSV genomic copies, and a significant reduction in the gross lung pathology (P < 0.05) was observed following PRRSV infection in rSPV-mp2-immunized animals. The results suggest that the recombinant rSPV-mp2 provided pigs with significant protection against PRRSV infection.

Comparison of homologous and heterologous prime-boost immunizations combining MVA-vectored and plant-derived VP2 as a strategy against IBDV.

Different immunogens such as subunit, DNA or live viral-vectored vaccines against Infectious Bursal Disease virus (IBDV) have been evaluated in the last years. However, the heterologous prime-boost approach using recombinant modified vaccinia Ankara virus (rMVA), which has shown promising results in both mammals and chickens, has not been tried against this pathogen yet. IBD is a highly contagious and immunosuppressive disease of poultry that affects mainly young chicks. It is caused by IBDV, a double-stranded RNA virus carrying its main antigenic epitopes on the capsid protein VP2. Our objective was to evaluate the immune response elicited by two heterologous prime-boost schemes combining an rMVA carrying the VP2 mature gene (rVP2) and a recombinant VP2 protein produced in Nicotiana benthamiana (pVP2), and to compare them with the performance of the homologous pVP2-pVP2 scheme usually used in our laboratory. The SPF chickens immunized with the three evaluated schemes elicited significantly higher anti-VP2 antibody titers (p<0.001) and seroneutralizing titers (p<0.05) and had less T-cell infiltration (p<0.001), histological damage (p<0.001) and IBDV particles (p<0.001) in their bursae of Fabricius when compared with control groups. No significant differences were found between both heterologous schemes and the homologous one. However, the rVP2-pVP2 scheme showed significantly higher anti-VP2 antibody titers than pVP2-rVP2 and a similar tendency was found in the seroneutralization assay. Conversely, pVP2-rVP2 had the best performance when evaluated through bursal parameters despite having a less potent humoral immune response. These findings suggest that the order in which rVP2 and pVP2 are combined can influence the immune response obtained. Besides, the lack of a strong humoral immune response did not lessen the ability to protect from IBDV challenge. Therefore, further research is needed to evaluate the mechanisms by which these immunogens are working in order to define the combination that performs better against IBDV.

Recombinant Marek's disease virus type 1 provides full protection against very virulent Marek's and infectious bursal disease viruses in chickens.

Marek's disease virus (MDV) is a preferred vector in the construction of recombinant vaccines. However, bivalent vaccine based on MDV that confers full protection against both very virulent Marek's and infectious bursal disease virus (IBDV) infections in chickens has not been produced. Here we developed a system utilizing overlapping fosmid DNAs transfection that rescues an MDV type 1 (MDV1) vaccine strain. Using this system, we inserted the IBDV VP2 gene at MDV1 genome sites UL41, US10 and US2. The VP2 protein was stably expressed in the recombinant MDV-infected cells and self-assembled into IBDV subviral particles. Insertion of the VP2 gene did not affect the replication phenotype of MDV in cell cultures, nor did it increase the virulence of the MDV vaccine strain in chickens. After challenge with very virulent IBDV, r814US2VP2 conferred full protection, whereas r814UL41VP2 and r814US10VP2 provided partial or no protection. All the three recombinant vaccines provided full protection against very virulent MDV challenge in chickens. These results demonstrated that r814US2VP2 could be used as a promising bivalent vaccine against both Marek's and infectious bursal diseases in chickens.

Effects of a broad-spectrum caspase inhibitor, Z-VAD(OMe)-FMK, on viral hemorrhagic septicemia virus (VHSV) infection-mediated apoptosis and viral replication.

In the development of inactivated or attenuated viral vaccines for cultured fish, viral titers harvested from the cultured cells would be the most important factor for the determination of vaccine's cost effectiveness. In this study, we hypothesized that the lengthening of cell survival time by the inhibition of apoptosis can lead to an increase of the final titer of viral hemorrhagic septicemia virus (VHSV). To test the hypothesis, we investigated the effects of a broad-spectrum caspase inhibitor, Z-VAD(OMe)-FMK, on VHSV infection-mediated apoptosis in Epithelioma papulosum cyprini (EPC) cells and on the VHSV titers. VHSV infection induced the DNA laddering in EPC cells, and the progression of DNA fragmentation was in proportion to the CPE extension. The progression of DNA fragmentation in EPC cells infected with VHSV was clearly inhibited by exposure to Z-VAD(OMe)-FMK, and the inhibition was intensified according to the increase of the inhibitor concentration. These results confirmed the previous reports that the death of host cells by VHSV infection is through apoptosis. Cells infected with a recombinant VHSV, rVHSV-ΔNV-eGFP, that was generated from our previous study by replacement of the NV gene ORF with the enhanced green fluorescent protein (eGFP) gene ORF, showed earlier and more distinct DNA fragmentations compared to the cells infected with wild-type VHSV, suggesting the inhibitory role of the NV protein in VHSV-mediated apoptosis that was previously reported. The final viral titers in the supernatant isolated from Z-VAD(OMe)-FMK treated cells after showing an extensive CPE were significantly higher than the viral titers from cells infected with virus alone, indicating that the delay of apoptosis by Z-VAD(OMe)-FMK extended the survival time of EPC cells, which lengthen the time for VHSV replication in the cells. In conclusion, Z-VAD(OMe)-FMK-mediated inhibition of apoptosis significantly increased the final titers of both wild-type VHSV and rVHSV-ΔNV-eGFP, indicating that apoptosis inhibition can be a way to get higher titers of VHSV.

Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer.

Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.

Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate.

UNLABELLED: Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the first and second leading viral agents of severe respiratory tract disease in infants and young children worldwide. Vaccines are not available, and an RSV vaccine is particularly needed. A live attenuated chimeric recombinant bovine/human PIV3 (rB/HPIV3) vector expressing the RSV fusion (F) glycoprotein from an added gene has been under development as a bivalent vaccine against RSV and HPIV3. Previous clinical evaluation of this vaccine candidate suggested that increased genetic stability and immunogenicity of the RSV F insert were needed. This was investigated in the present study. RSV F expression was enhanced 5-fold by codon optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypofusogenic phenotype that presumably reflects the original isolate. We then compared vectors expressing stabilized prefusion and postfusion versions of RSV F. In a hamster model, prefusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wild-type (wt) RSV challenge. In contrast, a vector expressing the postfusion F was less immunogenic and protective. The genetic stability of the RSV F insert was high and was not affected by enhanced expression or the prefusion or postfusion conformation of RSV F. These studies provide an improved version of the previously well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. IMPORTANCE: Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are two major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV. Previous clinical evaluation indicated the need to increase the immunogenicity and genetic stability of the RSV F insert. Here, we increased RSV F expression by codon optimization and by modifying the RSV F amino acid sequence to conform to that of an early passage of the original isolate. This resulted in a hypofusogenic phenotype, which likely represents the original phenotype before adaptation to cell culture. We also included stabilized versions of prefusion and postfusion RSV F protein. Prefusion RSV F induced a larger quantity and higher quality of RSV-neutralizing serum antibodies and was highly protective. This provides an improved candidate for further clinical evaluation.

Combined cytolytic effects of a vaccinia virus encoding a single chain trimer of MHC-I with a Tax-epitope and Tax-specific CTLs on HTLV-I-infected cells in a rat model.

Adult T cell leukemia (ATL) is a malignant lymphoproliferative disease caused by human T cell leukemia virus type I (HTLV-I). To develop an effective therapy against the disease, we have examined the oncolytic ability of an attenuated vaccinia virus (VV), LC16m8Δ (m8Δ), and an HTLV-I Tax-specific cytotoxic T lymphocyte (CTL) line, 4O1/C8, against an HTLV-I-infected rat T cell line, FPM1. Our results demonstrated that m8Δ was able to replicate in and lyse tumorigenic FPM1 cells but was incompetent to injure 4O1/C8 cells, suggesting the preferential cytolytic activity toward tumor cells. To further enhance the cytolysis of HTLV-I-infected cells, we modified m8Δ and obtained m8Δ/RT1AlSCTax180L, which can express a single chain trimer (SCT) of rat major histocompatibility complex class I with a Tax-epitope. Combined treatment with m8Δ/RT1AlSCTax180L and 4O1/C8 increased the cytolysis of FPM1V.EFGFP/8R cells, a CTL-resistant subclone of FPM1, compared with that using 4O1/C8 and m8Δ presenting an unrelated peptide, suggesting that the activation of 4O1/C8 by m8Δ/RT1AlSCTax180L further enhanced the killing of the tumorigenic HTLV-I-infected cells. Our results indicate that combined therapy of oncolytic VVs with SCTs and HTLV-I-specific CTLs may be effective for eradication of HTLV-I-infected cells, which evade from CTL lysis and potentially develop ATL.

The combination of attenuated Newcastle disease (ND) vaccine with rHVT-ND vaccine at 1 day old is more protective against ND virus challenge than when combined with inactivated ND vaccine.

The recurrent outbreaks of fatal Newcastle disease (ND) in commercial poultry flocks throughout the world indicate that routine vaccinations are failing to sufficiently induce the high levels of immunity necessary to control ND. There is a need for vaccination programmes that could be initiated at 1-day-old for mass application and which would induce a long-lasting immunity, with no need for a booster vaccination at a later age. In this context, the duration of immunity delivered by a vaccination programme including a recombinant herpesvirus of turkeys expressing the F gene of ND virus (rHVT-ND) and live ND vaccine at 1-day-old was compared with a classical programme that included a conventional live and an inactivated ND vaccine at the same age in commercial layer chickens. The humoral, cell-mediated and local immunity were followed weekly and birds were challenged with a viscerotropic velogenic ND virus strain at 6 and 10 weeks of age. We determined that immunity induced by the vaccination programme involving the rHVT-ND vaccine was more protective than that provided by the conventional vaccine-based regime. This might be related to a T-helper type 1 (Th1) cellular-driven immunological response, in contrast to the T-helper type 2 (Th2) humoral-oriented immune response provided by the current conventional vaccine-based vaccination programmes.

Heat-shock proteins as dendritic cell-targeting vaccines--getting warmer.

Heat-shock proteins (hsp) provide a natural link between innate and adaptive immune responses by combining the ideal properties of antigen carriage (chaperoning), targeting and activation of antigen-presenting cells (APC), including dendritic cells (DC). Targeting is achieved through binding of hsp to distinct cell surface receptors and is followed by antigen internalization, processing and presentation. An improved understanding of the interaction of hsp with DC has driven the development of numerous hsp-containing vaccines, designed to deliver antigens directly to DC. Studies in mice have shown that for cancers, such vaccines generate impressive immune responses and protection from tumour challenge. However, translation to human use, as for many experimental immunotherapies, has been slow partly because of the need to perform trials in patients with advanced cancers, where demonstration of efficacy is challenging. Recently, the properties of hsp have been used for development of prophylactic vaccines against infectious diseases including tuberculosis and meningitis. These hsp-based vaccines, in the form of pathogen-derived hsp-antigen complexes, or recombinant hsp combined with selected antigens in vitro, offer an innovative approach against challenging diseases where broad antigen coverage is critical.

Modification in media composition to obtain secretory production of STxB-based vaccines using Escherichia coli.

Shiga toxin B-subunit (STxB) from Shigella dysenteriae targets in vivo antigen to cancer cells, dendritic cells (DC) and B cells, which preferentially express the globotriaosylceramide (Gb3) receptor. This pivotal role has encouraged scientists to investigate fusing STxB with other clinical antigens. Due to the challenges of obtaining a functional soluble form of the recombinant STxB, such as formation of inclusion bodies during protein expression, scientists tend to combine STxB with vaccine candidates rather than using their genetically fused forms. In this work, we fused HPV16 E7 as a vaccine candidate to the recombinantly-produced STxB. To minimize the formation of inclusion bodies, we investigated a number of conditions during the expression procedure. Then various strategies were used in order to obtain high yield of soluble recombinant protein from E. coli which included the use of different host strains, reduction of cultivation temperature, as well as using different concentrations of IPTG and different additives (Glycin, Triton X-100, ZnCl(2)). Our study demonstrated the importance of optimizing incubation parameters for recombinant protein expression in E. coli; also showed that the secretion production can be achieved over the course of a few hours when using additives such as glycine and Triton X-100. Interestingly, it was shown that when the culture mediums were supplemented by additives, there was an inverse ratio between time of induction (TOI) and the level of secreted protein at lower temperatures. This study determines the optimal conditions for high yield soluble E7-STxB expression and subsequently facilitates reaching a functionally soluble form of STxB-based vaccines, which can be considered as a potent vaccine candidate for cervical cancer.

Modification of one epitope-flanking amino acid allows for the induction of friend retrovirus-specific CD8+ T cells by Adenovirus-based immunization.

While Friend retrovirus-infected mice readily mount a vigorous CD8(+) T cell response to the leader-gag-derived peptide GagL(85-93), no GagL(85-93)-specific T cells were detectable in mice immunized against Friend virus (FV) with viral vectors or DNA vaccines. By exchanging one epitope-flanking amino acid or using a scaffold protein we were able to demonstrate for the first time the induction of GagL(85-93)-specific CD8(+) T cells by genetic vaccination and show their high protective effect against FV challenge infection.

Novel AAV-based genetic vaccines encoding truncated dengue virus envelope proteins elicit humoral immune responses in mice.

The envelope protein of dengue virus is involved in host cell attachment for entry and induction of protective immunity. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we developed a genetic vaccine based on a novel adeno-associated viral (AAV) vector expressing the carboxy-terminal truncated envelope protein (79E) of dengue virus. The expression of the recombinant 79E protein in HEK 293 cells was confirmed by Western blot. Vectors packaged with novel AAV capsids (AAV2/8 or AAV2/rh32.33) were injected into C57BL/6 mice intramuscularly. Dengue virus antigen was produced in the mice and induced long-lasting antibody responses against the dengue virus still detectable 20 weeks after immunization. AAV2/8 vaccine induced higher anti-dengue virus antibody levels than AAV2/rh32.33 vaccine or AAV plasmid. Furthermore, the anti-dengue antibodies could neutralize homogeneous dengue virus. These results demonstrated that the AAV vaccines possessed appropriate immunogenicity and could be used for the development of an effective dengue vaccine.

Immunogenicity of a bovine herpes virus I peptide expressed in tandem copies in attenuated Salmonella.

A live system to release heterologous antigens using an attenuated Salmonella strain was developed. We transformed Salmonella typhimurium LVR03 (S. LVR03) with a recombinant pTECH2 vector encoding 0, 1, 2, and 4 tandem copies of an imunogenic peptide of bovine herpes virus-1 (BoHV-1) glycoprotein D (gD). The system used yielded peptides fused to the non-toxic C fragment of the tetanus toxin (TetC), which has been shown to have adjuvant properties. Inoculation of BALB/c mice with the transformed Salmonella strains gave rise to a mild self-limited infection, with primary replication of bacteria occurring in Peyer's patches, even when the bacteria was administered intranasally. Humoral and cellular immune responses directed against the BoHV-1 antigens were evaluated after oral or intranasal administration of the recombinant bacteria. The results showed that the S. LVR03-dimer vaccine induced specific humoral (IgG in serum and IgG(1) and IgA in saliva), and cellular immune responses (lymphoproliferation and lymphokine secretion), against not only the selected peptide and whole gD, but also against BoHV-1, when administered intranasally. This is the first time Salmonella has been used as an expression vector to induce immunity against BoHV-1. This work demonstrates the feasibility of using this antigen-release system and encourages future experimentation with a bovine experimental model.