Antigenicity and immunogenicity of recombinant proteins comprising African swine fever virus proteins p30 and p54 fused to a cell-penetrating peptide.

African swine fever (ASF) is a highly fatal swine disease threatening the global pig industry. Currently, vaccine is not commercially available for ASF. Hence, it is desirable to develop effective subunit vaccines against ASF. Here, we expressed and purified two recombinant fusion proteins comprising ASFV proteins p30 and p54 fused to a novel cell-penetrating peptide Z12, which were labeled as ZPM (Z12-p30-modified p54) and ZPMT (Z12-p30-modified p54-T cell epitope). Purified recombinant p30 and modified p54 expressed alone or fused served as controls. The transduction capacity of these recombinant proteins was assessed in RAW264.7 cells. Both ZPM and ZPMT exhibited higher transduction efficiency than the other proteins. Subsequently, humoral and cellular immune responses elicited by these proteins were evaluated in mice. ZPMT elicited the highest levels of antigen-specific IgG responses, cytokines (interleukin-2, interferon-γ, and tumor necrosis factor-α) and lymphocyte proliferation. Importantly, sera from mice immunized with ZPM or ZPMT neutralized greater than 85% of ASFV in vitro. Our results indicate that ZPMT induces potent neutralizing antibody responses and cellular immunity in mice. Therefore, ZPMT may be a suitable candidate to elicit immune responses in swine, providing valuable information for the development of subunit vaccines against ASF.

Characterizations of glucose-rich polysaccharides from Amomum longiligulare T.L. Wu fruits and their effects on immunogenicities of infectious bursal disease virus VP2 protein.

The aim of this study is to explore the characterization of Amomum longiligulare T.L. Wu fruits polysaccharide (ALP) and their immune enhancement effects. Two homogeneous polysaccharides (ALP1 and ALP2) were isolated from the fruits. The structural characterization results showed that ALP1 (26.10 kDa) and ALP2 (64.10 kDa) were both mainly composed of glucose. Furthermore, ALP1 was consisted of (1,2)-α-D-Glcp, (1,2,3)-α-D-Glcp and T-α-D-Glcp, while ALP2 was consisted of T-α-D-Glcp, (1,3)-α-D-Glcp and (1,3,6)-α-D-Glcp. Afterwards, the immune enhancement effects of two polysaccharides were evaluated by determining their effects on immunogenicities of infectious bursal disease virus (IBDV) VP2 protein. Chickens were immunized with IBDV VP2 protein accompanied with ALP1/ALP2. And the results indicated both ALP1 and ALP2 promoted the weights and bursa of fabricius indexes of chickens. In addition, both two polysaccharides increased specific IBDV antibody levels, while ALP1 possessed higher immune enhancement ability and was expected to be an adjuvant for IBDV VP2 protein.

Adaptation of a live-attenuated genotype I Japanese encephalitis virus to vero cells is associated with mutations in structural protein genes.

The SD12-F120 is a live-attenuated genotype I strain of Japanese encephalitis virus (JEV) and was obtained by serial passage of wild-type strain SD12 on BHK-21 cells combined with multiple plaque purification and virulence selection in mice. The large scale production and vast clinical trials always demand ideal safety and efficacy profile of live-attenuated vaccines. In the present study, SD12-F120VC has undergone serial passaging of P1-P30 in WHO qualified Vero cells to assess the potential effect of adaptation to growth on Vero cells. The series of experiments showed that vaccine SD12-F120VC (Vero cell adapted) variants have consistently increased in peak virus titer compared to early passages and have good adaptation to growth in Vero cells. The animal experiments showed that Vero cell adapted SD12-F120VC variants have attenuation phenotype in suckling mice and the plaque morphology for all SD12-F120VC variants was small. Vaccination of mice with SD12-F120VC vaccine produced complete protection for homologous SD12 genotype I strain, but failed to give the complete protection of vaccinated mice against the challenge of heterologous N28 genotype III strain. In response to immunization of SD12-F120VC in mice, the neutralizing antibodies titer against homologous SD12-F120VC and SD12 (GI) was higher than heterologous N28 (GIII) strain. The prM protein has 6 amino acid substitutions, of which 5 amino acid changes were confined at the start of the pr domain in the ∼40 amino acids, and some mutations in the pr domain of prM might contribute to Vero cell adaptation. Our findings in this study are important for validation, evaluation and quality control study of live attenuated flaviviruses vaccines and show that Vero cells are a suitable substrate for the production of a safe and stable live-attenuated JEV vaccine.

Superior adjuvanticity of the genetically fused D1 domain of Neisseria meningitides Ag473 lipoprotein among three Toll-like receptor ligands.

Toll-like receptor (TLR) ligands have emerged as the attractive adjuvant for subunit vaccines. However, selection of TLR ligands needs to be rationally chosen on the basis of antigen and adjuvant properties. In the present study, we expressed the Ag473 lipoprotein from Neisseria meningitides, flagellin FlaB from Vibrio vulnificus and heat shock protein 70 from Mycobacterium tuberculosis (mHsp70) in Escherichia coli as single proteins and fusion proteins with VP2 protein of infectious bursal disease virus (IBDV). Both cellular and humoral adjuvanticities of the three TLR ligands were compared by immunization of mice in two different ways. Among the three co-administered TLR ligands, recombinant Ag473 lipoprotein exhibited the highest cellular and humoral adjuvanticities, including promotion of IL-4, IL-12, IFN-γ and IBDV VP2-specific antibody production. Among the three genetically fused TLR ligands, fusion with Ag473 D1 domain exhibited the highest cellular and humoral adjuvanticities. Overall, the adjuvanticities of genetically fused TRL ligands were significantly higher than that of co-administered TLR ligands. Fusion with Ag473 D1 domain exhibited superior adjuvanticity among the three TLR ligands delivered in two different ways.

Oral immunization with recombinant Lactobacillus casei displayed AHA1-CK6 and VP2 induces protection against infectious pancreatic necrosis in rainbow trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV) primarily infects larvae and young salmonid with serious economic losses, which causes haemorrhage and putrescence of hepatopancreas. To develop a more effective oral vaccine against IPNV infection, the aeromonas hydrophila adhesion (AHA1) gene was used as a targeting molecule for intestinal epithelial cells. A genetically engineered Lactobacillus casei (pPG-612-AHA1-CK6-VP2/L. casei 393) was constructed to express the AHA1-CK6-VP2 fusion protein. The expression of interest protein was confirmed by western blotting and the immunogenicity of pPG-612-AHA1-CK6-VP2/L. casei 393 was evaluated. And the results showed that more pPG-612-AHA1-CK6-VP2/L. casei 393 were found in the intestinal mucosal surface of the immunized group. The Lactobacillus-derived AHA1-CK6-VP2 fusion protein could induce the production of serum IgM and skin mucus IgT specific for IPNV with neutralizing activity in rainbow trouts. The levels of IL-1ß, IL-8 and TNF-α isolated from the lymphocytes stimulated by AHA1-CK6-EGFP produced were significantly higher than EGFP group. For transcription levels of IL-1ß, IL-8, CK6, MHC-II, Mx and TNF-1α in the spleen, the result indicated that the adhesion and target chemokine recruit more immune cells to induce cellular immunity. The level of IPNV in the immunized group of pPG-612-AHA1-CK6-VP2/L. casei 393 was significantly lower than that in the control groups. These data indicated that the adhesion and target chemokine could enhance antigen delivery efficiency, which provides a valuable strategy for the development of IPNV recombination Lactobacillus casei oral vaccine in the future.

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.

Evaluation of two strains of Marek's disease virus serotype 1 for the development of recombinant vaccines against very virulent infectious bursal disease virus.

Attenuated strains of Marek's disease virus serotype 1 (MDV1), and the closely related herpesvirus of turkeys, are among the most potent vectors for development of recombinant vaccines for poultry. To investigate the effects of MDV1 strain characteristics on the protective efficacy of the recombinant vaccines, we developed two recombinant MDV1 vaccines for expressing the VP2 gene of infectious bursal disease virus (IBDV) based on two different MDV1 strains, the attenuated strain 814 and the Meq gene-deleted recombinant MDV1 strain rLMS△Meq, as the viral vectors. The r814-VP2 virus based on the 814 strain exhibited higher replication efficiency in cell culture while lower viral titers in chickens, compared to rLMS△Meq-VP2 derived from the rLMS△Meq strain. Further studies indicated that r814-VP2 produced higher levels of VP2 protein in cells and elicited stronger immune responses against IBDV in chickens than rLMS△Meq-VP2. After IBDV challenge, rLMS△Meq-VP2 provided 50% protection against mortality, and the birds that survived developed bursal atrophy and gross lesions. In contrast, r814-VP2 conferred complete protection not only against development of clinical signs and mortality, but also against the formation of bursal lesions. The results indicate that different MDV1 vector influences the protective efficacy of recombinant MDV1 vaccines. The r814-VP2 has the potential to serve as a bivalent vaccine against two important lethal pathogens of chickens.

A novel poxvirus-based vaccine, MVA-CHIKV, is highly immunogenic and protects mice against chikungunya infection.

UNLABELLED: There is a need to develop a single and highly effective vaccine against the emerging chikungunya virus (CHIKV), which causes a severe disease in humans. Here, we have generated and characterized the immunogenicity profile and the efficacy of a novel CHIKV vaccine candidate based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the CHIKV C, E3, E2, 6K, and E1 structural genes (termed MVA-CHIKV). MVA-CHIKV was stable in cell culture, expressed the CHIKV structural proteins, and triggered the cytoplasmic accumulation of Golgi apparatus-derived membranes in infected human cells. Furthermore, MVA-CHIKV elicited robust innate immune responses in human macrophages and monocyte-derived dendritic cells, with production of beta interferon (IFN-ß), proinflammatory cytokines, and chemokines. After immunization of C57BL/6 mice with a homologous protocol (MVA-CHIKV/MVA-CHIKV), strong, broad, polyfunctional, and durable CHIKV-specific CD8(+) T cell responses were elicited. The CHIKV-specific CD8(+) T cells were preferentially directed against E1 and E2 proteins and, to a lesser extent, against C protein. CHIKV-specific CD8(+) memory T cells of a mainly effector memory phenotype were also induced. The humoral arm of the immune system was significantly induced, as MVA-CHIKV elicited high titers of neutralizing antibodies against CHIKV. Remarkably, a single dose of MVA-CHIKV protected all mice after a high-dose challenge with CHIKV. In summary, MVA-CHIKV is an effective vaccine against chikungunya virus infection that induced strong, broad, highly polyfunctional, and long-lasting CHIKV-specific CD8(+) T cell responses, together with neutralizing antibodies against CHIKV. These results support the consideration of MVA-CHIKV as a potential vaccine candidate against CHIKV. IMPORTANCE: We have developed a novel vaccine candidate against chikungunya virus (CHIKV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing the CHIKV C, E3, E2, 6K, and E1 structural genes (termed MVA-CHIKV). Our findings revealed that MVA-CHIKV is a highly effective vaccine against chikungunya virus, with a single dose of the vaccine protecting all mice after a high-dose challenge with CHIKV. Furthermore, MVA-CHIKV is highly immunogenic, inducing strong innate responses: high, broad, polyfunctional, and long-lasting CHIKV-specific CD8(+) T cell responses, together with neutralizing antibodies against CHIKV. This work provides a potential vaccine candidate against CHIKV.

Increasing versatility of the DNA vaccines through modification of the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells.

The route of administration of DNA vaccines can play a key role in the magnitude and quality of the immune response triggered after their administration. DNA vaccines containing the gene of the membrane-anchored glycoprotein (gpG) of the fish rhabdoviruses infectious haematopoietic necrosis virus (IHNV) or viral haematopoietic septicaemia virus (VHSV), perhaps the most effective DNA vaccines generated so far, confer maximum protection when injected intramuscularly in contrast to their low efficacy when injected intraperitoneally. In this work, taking as a model the DNA vaccine against VHSV, we focused on developing a more versatile DNA vaccine capable of inducing protective immunity regardless of the administration route used. For that, we designed two alternative constructs to gpG1₋507 (the wild type membrane-anchored gpG of VHSV) encoding either a soluble (gpG1₋462) or a secreted soluble (gpG(LmPle20-462)) form of the VHSV-gpG. In vivo immunisation/challenge assays showed that only gpG(LmPle20-462) (the secreted soluble form) conferred protective immunity against VHSV lethal challenge via both intramuscular and intraperitoneal injection, being this the first description of a fish viral DNA vaccine that confers protection when administered intraperitoneally. Moreover, this new DNA vaccine construct also conferred protection when administered in the presence of an oil adjuvant suggesting that DNA vaccines against rhabdoviruses could be included in the formulation of current multicomponent-intaperitoneally injectable fish vaccines formulated with an oil adjuvant. On the other hand, a strong recruitment of membrane immunoglobulin expressing B cells, mainly membrane IgT, as well as t-bet expressing T cells, at early times post-immunisation, was specifically observed in the fish immunised with the secreted soluble form of the VHSV-gpG protein; this may indicate that the subcellular location of plasmid-encoded antigen expression in the in vivo transfected cells could be an important factor in determining the ways in which DNA vaccines prime the immune response.

Recent advances in mucosal immunization using virus-like particles.

Mucosal immunization offers the promises of eliciting a systemic and mucosal immune response, as well as enhanced patient compliance. Mucosal vaccination using defined antigens such as proteins and peptides requires delivery systems that combine good safety profiles with strong immunogenicity, which may be provided by virus-like particles (VLP). VLP are assembled from viral structural proteins and thus are devoid of any genetic material. They excel by mimicking natural pathogens, therefore providing antigen-protecting particulate nature, inherent immune-cell stimulatory mechanisms, and tissue-specific targeting depending on their parental virus. Nevertheless, despite of promising preclinical results, VLP remain rarely investigated in clinical studies. This review is intended to give an overview of obstacles and promises of VLP-based mucosal immunization as well as to identify strategies to further improve VLP while maintaining a good safety and tolerability profile.

DNA vaccination with a gene encoding VP60 elicited protective immunity against rabbit hemorrhagic disease virus.

Rabbit hemorrhagic disease (RHD) is a contagious disease in adult rabbits, with high mortality, that occurs throughout the world. The VP60 protein has been implicated as main protein antigen in virus diagnosis and vaccine design. In this report, we describe the construction of a novel DNA vaccine (pcDNA-VP60) expressing the RHDV capsid protein (VP60), and the expression of the recombinant protein was identified through indirect immunofluorescence assay (IFA) and Western blot assay. VP60 protein self-assembled to form virus-like particles (VLPs) observed by electron microscopy were morphologically similar to native virions. For the evaluation of vaccine efficacy, rabbits were inoculated with PBS, pcDNA3.1((+)), pcDNA-VP60 or RHDV inactive vaccine. They were challenged with RHDV-TP isolate four weeks after last boost immunization. In all cases, the rabbits immunized with pcDNA-VP60 developed high level of RHDV-specific antibodies and cellular immune response. The rabbits injected with DNA vaccine were completely protected against RHDV challenge like commercial RHDV inactive vaccine, moreover, RHDV viral load was significantly reduced in the liver samples from immunized rabbits. The recombinant DNA vaccine may provide a novel strategy for the immunization of rabbits for the control of RHDV.

Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV) in cattle.

BACKGROUND: Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen. FINDINGS: Replicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and expression was confirmed in vitro using polyclonal and monoclonal antibodies specific to E2. Vaccine made from particles was generated in Vero cells and administered to BVDV free calves in a prime/boost regimen at two dosage levels. Vaccination resulted in neutralizing antibody titers that cross-neutralized both type 1 and type 2 BVD genotypes following booster vaccination. Additionally, high dose vaccine administration demonstrated some protection from clinical disease and significantly reduced the degree of leukopenia caused by viral infection. CONCLUSIONS: Replicon particle vaccines administered in a prime/boost regimen expressing BVDV E2 glycoprotein can induce cross-neutralizing titers, reduce leukopenia post challenge, and mitigate clinical disease in calves. This strategy holds promise for a safe and effective vaccine to BVDV.

Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice.

Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni-NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.

VP22 and cytosine deaminase fusion gene modified tissue-engineered neural stem cells for glioma therapy.

PURPOSE: The herpes simplex virus type 1 tegument protein VP22 has the remarkable property of intercellular trafficking, thus making it a promising tool for improving gene transfer efficiency. METHODS: To investigate whether the fusion of VP22 to the cytosine deaminase (CD) suicide gene could enhance the therapeutic efficiency of neural stem cells (NSCs) in the treatment for C6 glioma, the lentiviral vectors pHIV-VP(22)-EGFP, pHIV-CD, and pHIV-VP(22)-CD were constructed based on the pHIV-EGFP vector. After packaging, vectors were transduced into rat NSCs. RESULTS: Fluorescence-activated cell sorting analysis revealed that the fusion of VP22-EGFP increased the expression rate of EGFP in NSCs compared with lenti-EGFP transduced cells. Under incubation with the prodrug 5-fluorocytosine (5-FC), the survival rates of C6 cells co-cultured with NSCs/VP(22)-CD (NSCs transduced with lenti-VP(22)-CD) decreased tremendously compared with those of C6 and NSCs/CD. Similar results were also observed in vivo; a significant reduction in tumor volumes in C6 glioma-bearing rats was observed in the NSCs/VP(22)-CD therapy group when compared with other control groups. CONCLUSIONS: Our results reveal that VP22 increases the transduction efficiency of lentivirus into NSCs and enhances the therapeutic efficacy of CD-engineered rat NSCs in the treatment for C6 glioma, demonstrating that VP22 might be a useful tool for the gene therapy of engineered NSCs and providing a potential novel strategy for enhancing the effectiveness of gene therapy in other diseases.

DNA-vaccine platform development against H1N1 subtype of swine influenza A viruses.

Swine influenza virus (SIV) is an important viral pathogen in pig populations. However, commercial vaccines cannot provide complete protection with induced humoral immunity only, and require frequent updates to fight against current isolates. DNA vaccination is an effective means of eliciting both arms of the immune system, the humoral and cellular immune responses. In this study, DNA vector pcDNA3.1 was inserted with a chimeric intron downstream of the CMV promoter region followed by a Kozak sequence to enhance the expression of gene inserts. The C-terminal of the VP22 gene (VP22c), encoding the tegument protein of bovine herpesvirus-1, was fused separately to the N-terminal of four quadruplicated epitopes: two B-cell epitopes (HA91-108 and M2e), and two T-cell epitopes (NP366-374 and NP380-393), which were conserved, at least among the three SIV subtypes prevailing in pig populations in North America. Linker -KK- was used to space between each copy of the two B-cell epitopes, and -RVKR- was used for the two T-cell epitopes, in order to enhance the presentation of epitopes to the immune system. The expression of epitopes was confirmed in in vitro transfection of 293FT cells, and higher percentages of epitope-positive cells were achieved from the plasmids containing VP22c than those without. After the DNA plasmids were administered to mice intramuscularly in combination or separately, or boosted with recombinant proteins of quadruplicated epitopes fused to VP22c, the vaccine stimulated the desired epitope-specific humoral immunity to the two B-cell epitopes, and cellular immunity to the epitope NP380-393. Our results indicate that plasmids with quadruplicated epitopes fused to the VP22c may be a potential vehicle in developing epitopes as vaccines against SIV.

Recent advances of cowpea mosaic virus-based particle technology.

Particles of cowpea mosaic virus (CPMV) have enjoyed considerable success as a means of presenting peptides for vaccine purposes. However, the existing technology has limitations in regard to the size and nature of the peptides which can be presented and has problems regarding bio-containment. Recent developments suggest ways by which these problems can be overcome, increasing the range of potential applications of CPMV-based particle technology.

Study of a chimeric foot-and-mouth disease virus DNA vaccine containing structural genes of serotype O in a genome backbone of serotype Asia 1 in guinea pigs.

Since foot-and-mouth disease virus (FMDV) serotypes display a great genetic and antigenic diversity, there is a constant requirement to monitor the performance of FMDV vaccines in the field with respect to their antigenic coverage. To avoid possible antigenic changes in field FMDV isolates during their adaptation to BHK-21 cells, a standard step used in production of conventional FMDV vaccines, the custom-made chimeric conventional or DNA vaccines, in which antigenic determinants are replaced with those of appropriate field strains, should be constructed. Using this approach, we made a plasmid-based chimeric FMDV DNA vaccine containing structural genes of serotype O in the genome backbone of serotype Asia 1, all under the control of Human cytomegalovirus (HCMV) immediate early gene promoter. BHK-21 cells transfected with the chimeric DNA vaccine did not show cytopathic effect (CPE), but expressed virus-specific proteins as demonstrated by 35S-methionine labeling and immunoprecipitation. Guinea pigs immunized with the chimeric DNA vaccine produced virus-specific antibodies assayed by ELISA and virus neutralization test (VNT), respectively. The chimeric DNA vaccine showed a partial protection of guinea pigs challenged with the virulent FMDV. Although the chimeric DNA vaccine, in general, was not as effective as a conventional one, this study encourages further work towards the development of genetically engineered custom-made chimeric vaccines against FMDV.

Immunogenicity properties of authentic and heterologously synthesized structural protein VP2 of infectious pancreatic necrosis virus.

The sole coat protein VP2 of infectious pancreatic necrosis virus (IPNV) was isolated and purified from intact virions, propagated in CHSE-214 cells. Likewise was the full-length VP2 protein isolated and purified upon cloning and expression of the corresponding complete gene in E. coli. The two purified proteins of different synthetic origins carrying identical primary structures were utilized in an immunization program using a rabbit model. Sera obtained against both immunogens react equally well with authentic and recombinant VP2 in Western blots and ELISAs. Also, the total net binding forces as determined by avidity index (AI) calculations were high and of similar stature, exceeding 80. An IPNV infection of susceptible and permissive CHSE-214 cells could only be neutralized by IgG preparations obtained from rabbits immunized with authentic VP2. Only such antibodies were able to aggregate and sediment radiolabeled virions in glycerol gradients upon rate zonal centrifugations. The presence of sugar moieties on the authentic protein is suggested to be of pivotal importance in eliciting an immune response capable of preventing infection in cell cultures in vitro.

Protective cell-mediated immunity by DNA vaccination against Papillomavirus L1 capsid protein in the Cottontail Rabbit Papillomavirus model.

Papillomavirus major capsid protein L1 has successfully stimulated protective immunity against virus infection by induction of neutralizing antibodies in animal models and in clinical trials. However, the potential impact of L1-induced protective cell-mediated immune (CMI) responses is difficult to measure in vivo because of the coincidence of anti-L1 antibody. In this study, we tested the hypothesis that L1 could activate CMI, using the Cottontail Rabbit Papillomavirus (CRPV)-rabbit model. A unique property of this model is that infections can be initiated with viral DNA, thus bypassing all contributions to protection via neutralizing anti-L1 antibody. DNA vaccines containing either CRPV L1, or subfragments of L1 (amino-terminal two-thirds of L1 [L1N] and the carboxylterminal two-thirds of L1 [L1C]), were delivered intracutaneously into rabbits, using a gene gun. After three booster immunizations, the rabbits were challenged with several viral DNA constructs: wild-type CRPV, CRPV L1ATGko (an L1 ATG knockout mutation), and CRPV-ROPV hybrid (CRPV with a replacement L1 from Rabbit Oral Papillomavirus). Challenge of L1 DNA-vaccinated rabbits with wild-type CRPV resulted in significantly fewer papillomas when compared with challenge with CRPV L1ATGko DNA. Significantly smaller papillomas were found in CRPV L1-, L1N-, and L1C-vaccinated rabbits. In addition, rabbits vaccinated with either L1 or L1N grew significantly fewer and smaller papillomas when challenged with CRPV-ROPV hybrid DNA. Therefore, CRPV L1 DNA vaccination induced CMI responses to CRPV DNA infections that can contribute to protective immunity. Cross-protective immunity against CRPV L1 and ROPV L1 was elicited in these CRPV L1- and subfragment-vaccinated rabbits.

The recombinant rabbit hemorrhagic disease virus VP60 protein obtained from Pichia pastoris induces a strong humoral and cell-mediated immune response following intranasal immunization in mice.

Rabbit hemorrhagic disease (RHD) is a contagious and highly lethal viral disease of rabbits that spreads rapidly and infects animals by nasal, conjunctival and oral routes. Therefore, this experiment was undertaken to study the immune response generated after intranasal (i.n.) vaccination with the recombinant VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) expressed at high levels in Pichia pastoris. Groups of BALB/c mice were immunized with three doses of purified VP60 protein (Group 1), VP60 formulated within the cell debris fraction of the transformed yeast (Group 2) and placebo (Group 3) by intranasal route. Mice were also intramuscularly injected with purified VP60 protein (Group 4). A rapid antibody response specific against rabbit hemorrhagic disease virus was observed in all the experimental groups, except in Group 3, as detected by ELISA. The highest titers were found 60 days after the first immunization. Mice from Group 1 showed the highest IgG response (p<0.05) and the most balanced profile of IgG1, IgG2a and IgG2b subclasses. IgA titers specific to the virus were found only in animals from this group, which also developed the highest specific lymphocyte proliferative response. Interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) gene expression was also detected after an ex vivo-specific stimulation of mice from Groups 1 and 4. These data demonstrated the capacity of VP60 protein expressed in P. pastoris to elicit a potent humoral and cell-mediated immune response following an intranasal immunization scheme.