Hemagglutinin Displayed Baculovirus Protects Against Highly Pathogenic Influenza

Baculovirus (BV) replicating in insect cells can express a foreign gene product as part of its genome. The influenza hemagglutinin (HA) can be expressed from BV and displayed on the surface of baculovirus (HA-DBV). In this study we first generated six recombinant baculoviruses that expressed chimeric HAs with segments of the BV glycoprotein (gp64). The signal peptide (SP) and cytoplasmic tail (CT) domains of gp64 can enhance the display of HA from A/PR8/34 on BV surface, while the transmembrane (TM) domain of gp64 impairs HA display. Different doses of either live or β-propiolactone (BPL)-inactivated HA-DBV were administered to BALB/c mice. Live HA-DBV elicited higher hemagglutination-inhibition (HAI) titers than BPL-inactivated HA-DBV, and provided sterilizing protection. A second generation recombinant BV simultaneously displaying four HAs derived from four subclades of H5N1 influenza viruses was constructed. This tetravalent H5N1 HA-DBV vaccine elicited HAI titers against all four homologous H5N1 viruses, significantly decreasing viral lung titers of challenged mice and providing 100% protection against lethal doses of homologous H5N1 viruses. Moreover, mice vaccinated with HA-DBV had high levels of IFNγ-secreting and HA-specific CD8+ T cells. Taken together, this study demonstrates that HA-DBV can stimulate strong humoral, as well as cellular immune responses, and is an effective vaccine candidate for influenza.     

Is there an advantage to including the nucleoprotein in a rabies glycoprotein subunit vaccine?

The PV rabies (genotype 1) G and N proteins were produced by recombinant baculoviruses in insect cells. We tested the ability of recombinant antigens to synergistically induce an immune response and, particularly, to broaden the spectrum of Lyssavirus-neutralizing antibodies produced. Cell-free preparations of recombinant proteins caused an immune response. Recombinant rabies G protein (RRG) from infected cell extract or supernatant induced virus neutralizing antibodies (VNAb) against rabies (CVS virus (genotype 1) and in a less extent against European Bat Lyssavirus-1 (EBL-1:genotype 5) Recombinant rabies N protein (RRN) induced antibodies that reacted with the rabies virus ribonucleoprotein (RNP) and primed mice for both the production of VNAb induced by inactivated and purified rabies virus and the protection conferred by RNP. RRN also had an adjuvant effect on VNAb production induced by RRG when the two recombinant proteins were physically associated either encapsulated in liposomes or subjected to ultrasound treatment. However, there was no increase in production of VNAb directed against EBL-1 although classical vaccines (genotype 1) induce partial protection against this virus. thus, beside its adjuvant effect there is some doubt as to whether including rabies N protein in a rabies subunit vaccine containing the recombinant G protein would be advantageous.     

A novel bi-functional DNA vaccine expressing VP1 protein and producing antisense RNA targeted to 5'UTR of foot-and-mouth disease virus can induce both rapid inhibitory effect and specific immune response in mice

To overcome the inability of a conventional vaccine against a virus to induce rapid protection against viral challenge, a novel strategy was performed to generate a novel bi-functional vector expressing antisense RNA targeted to 5' untranslated regions (UTR) and VP1 protein of foot-and-mouth disease virus (FMDV). FMDV 5'UTR containing the viral RNA replication start elements was inserted inversely into the pIRES vector to produce antisense RNA, followed by insertion of FMDV VP1 gene to generate a recombinant plasmid pAS-IR-VP1. BHK-21 cells transfected with pAS-IR-VP1 plasmid showed a specific resistance against FMDV infection. In mice vaccinated with this plasmid, T cell proliferation was significantly higher than that in an unvaccinated control group. Anti-FMDV antibodies were detected up to 1:64 in the serum collected from mice boosted with pAS-IR-VP1 at 21 days after the first immunization. At 6h post-vaccination 50-83% of the suckling mice survived a challenge with FMDV. The results demonstrated that a novel bi-functional DNA vaccine, producing antisense RNA targeted to FMDV 5'UTR and expressing VP1 protein, has been successfully constructed and was able to induce a rapid inhibitory effect and immune response against FMDV infection in mice.     

A canine adenovirus type 2 vaccine vector confers protection against foot-and-mouth disease in guinea pigs

Vaccination is a key element in the control of foot-and-mouth disease (FMD). The majority of the antigenic sites that induce protective immune responses are localized on the FMD virus (FMDV) capsid that is formed by four virus-encoded structural proteins, VP1 to VP4. In the present study, recombinant canine adenovirus type 2 (CAV2)-based FMD vaccines, Cav-P1/3C R° and Cav-VP1 R°, respectively expressing the structural P1 precursor protein along with the non-structural 3C protein or expressing the structural VP1 protein of the FMDV strain O/FRA/1/2001, were evaluated as novel vaccines against FMD. A strong humoral immune response was elicited in guinea pigs (GP) following immunization with Cav-P1/3C R°, while administration of Cav-VP1 R° did not induce a satisfying antibody response in GP or mice. GP were then used as an experimental model for the determination of the protection afforded by the Cav-P1/3C R° vaccine against challenge with the FMDV strain O₁ Manisa/Turkey/1969. The Cav-P1/3C R° vaccine protected GP from generalized FMD to a similar extent as a high potency double-oil emulsion O₁ Manisa vaccine. The results of the present study show that CAV2-based vector vaccines can express immunogenic FMDV antigens and offer protection against generalized FMD in GP. This suggest that Cav-P1/3C R° FMDV vaccine may protect natural host species from FMD. In combination with an appropriate diagnostic test, the Cav-P1/3C R° FMDV vaccine may also serve as a marker vaccine to differentiate vaccinated from infected animals.     

Mucosal and systemic immune responses induced after oral delivery of vaccinia virus recombinants

The immune responses elicited after oral delivery of vaccinia virus (VV) recombinants are not well defined. In this study we show with mice, that after oral administration of a VV recombinant expressing the luciferase reporter gene, VV gene expression takes place for several days in gut-associated lymphoid (GALT) tissues as well as in the spleen. After 14 days, a significant mucosal IgA response against VV was detected in vaginal and intestinal washings, as well as a systemic specific IgG response, which was principally of the IgG2a subclass. Furthermore, orally immunized mice developed cellular immune responses to VV (CD8+ T cells and T helper activities) in mesenteric lymph nodes (MLN) and spleen. Oral immunization with a VV recombinant expressing, either the envelope protein of HIV or beta-galactosidase, induced a specific immune response, locally and systemically, against gp120 and beta-gal. The cytokine pattern found in supernatants of spleen and MLN cells after stimulation with VV antigens or gp120 was clearly of type 1 cytokines. These studies demonstrate that VV recombinants administered by the oral route generate mucosal and systemic immune responses against antigens of the virus vector and to the recombinant products. These observations are of significance in the use of poxvirus vectors as vaccines.     

Orally delivered foot-and-mouth disease virus capsid protomer vaccine displayed on T4 bacteriophage surface: 100% protection from potency challenge in mice

An orally delivered foot-and-mouth disease (FMD) vaccine has not previously been reported. By using a T4 bacteriophage nanoparticle surface gene-protein display system (T4-S-GPDS), we created a foot-and-mouth disease virus (FMDV) entire capsid protein vaccine candidate. On the T4 phage surface SOC site, a full length FMDV capsid precursor polyprotein (P1, 755 aa) and proteinase 3C (213 aa) derived from an infected pig of serotype O strain GD-10 (1999), were separately displayed on different T4 phage particle surfaces through inserting their coding region DNAs into the T4 phage genome, yielding phage strains T4-P1 and T4-3C. We also constructed a series of FMDV sub-full length capsid structural protein (subunit) containing T4 phage recombinant vaccines. Both sucking and young BALB/c mice were used as two kinds of FMDV vaccine potency evaluation models. Many groups of both model mice were vaccinated orally or by subcutaneous injection with varying FMDV-T4 phage recombinant vaccines, with and without addition of adjuvant, then challenged with a lethal dose of cattle source virulent FMDV. In the case of immunization with a mixture of phage T4-P1 and phage T4-3C particles without any adjuvant added, all mice were 100% protected following either oral or injection immunization, whereas 100% of the control, non-immunized mice and mice immunized with only T4 phage vector Z1/Zh(-) or wild-type T4(+)D phage died; in contrast, with FMDV subunit vaccine, less than 75% protection followed the same potency challenge in both mice model groups. In addition, two pigs immunized with a phage T4-P1 and phage T4-3C mix were protected upon housing together with infected pigs. This study represents a clear example of how FMD and other pathogenic disease vaccines can be prepared by a simple and efficient bacteriophage route.     

Characterization of recombinant foot-and-mouth disease virus pentamer-like structures expressed by baculovirus and their use as diagnostic antigens in a blocking ELISA

Non-infectious recombinant pentamer-like structures of the foot-and-mouth disease virus (FMDV) were expressed by baculovirus, and the antigenicity and immunogenicity of the proteins were analyzed in a blocking ELISA for the detection of FMDV antibodies. The recombinant pentamer-like structures were produced in insect (Sf9) cells that were inoculated with recombinant baculoviruses that expressed, simultaneously, the genes for the P1 and 3C proteins of FMDV from individual promoters. The FMDV pentamer-like structures were processed by viral 3C protease, as shown in Western blots, and were antigenic, as revealed by their reactivities in an indirect ELISA. Analysis by CsCl gradient centrifugation showed that the pentamer-like structures were similar to authentic pentameric subunits from FMDV in terms of sedimentation velocity. Furthermore, the pentamer-like structures induced high levels of FMDV-specific antibodies in mice following immunization. Observations made under the electron microscope revealed that the pentamer-like structures expressed by insect cells self-assembled to form pentameric subunits of 7-8 nm in diameter, which resemble the authentic FMDV (23+/-2 nm in diameter). The results indicate that these pentamer-like structures are as antigenic and immunogenic as authentic FMDV, although the former are smaller in size. Based on these results, a blocking ELISA was developed using the recombinant pentamer-like structure. The ELISA showed specificity of 99.5% and sensitivity of 98.5% when tested with FMDV antibody-negative and -positive sera, respectively. This blocking ELISA is highly specific and offers many advantages over the current ELISAs that use inactivated FMDV antigen. This is the first report of the production and diagnostic application of recombinant pentameric subunits of FMDV.     

Baculovirus surface display of sigmaC and sigmaB proteins of avian reovirus and immunogenicity of the displayed proteins in a mouse model

Avian reovirus (ARV), an important pathogen in poultry, causes arthritis, chronic respiratory disease, and malabsorption syndrome that cause considerable economic losses to the poultry industry. In present study, we have succeeded in construction of a universal baculovirus surface display system (UBSDS) that can display different foreign proteins on the envelope of baculovirus. Sequences encoding the signal peptide (SS), transmembrane domain (TM), and cytoplasmic domain (CTD) derived from the gp64 protein of baculovirus and histidine tag, respectively were inserted into the pBacCE vector. Four restriction enzyme sites between the histidine tag and gp64 transmembrane domain were established for expression of different foreign proteins. The transmembrane domain and CTD of gp64 in the platform were designed in order to improve stability and quantity of foreign proteins on the envelope of baculovirus. The sigmaC and sigmaB proteins of ARV are known to elicit neutralizing antibodies against ARV. The UBSDS was therefore used to express sigmaC and sigmaB proteins on the envelope of baculovirus. Two recombinant baculoviruses BacSC-sigmaC and BacSC-sigmaB have been successfully constructed. After infection, both His6-tagged recombinant sigmaC (rsigmaC) and sigmaB (rsigmaB) proteins were displayed on the envelope of recombinant baculoviruses and the recombinant viral proteins were anchored on the plasma membrane of Sf-9 cells, as revealed by immunofluorescence staining (IFS) and confocal microscopy. The antigenicity of rsigmaC and rsigmaB proteins was demonstrated by Western blotting assay. Immunogold electron microscopy demonstrated that both recombinant viruses displayed rsigmaC and rsigmaB proteins on the viral surface. Immunization of BALB/c mice with recombinant viruses, demonstrated that serum from the BacSC-sigmaC and BacSC-sigmaB treated models had significant higher levels of virus neutralization activities than the control groups. This demonstrates that the recombinant baculoviruses BacSC-sigmaC and BacSC-sigmaB can be a potential vaccine against ARV infections.     

Evaluation of Venezuelan Equine Encephalitis (VEE) replicon-based Outer surface protein A (OspA) vaccines in a tick challenge mouse model of Lyme disease

Venezuelan Equine Encephalitis (VEE) virus replicon particles (VRPs) encoding Borrelia burgdorferi Outer surface protein A (OspA) were evaluated for their ability to induce an immune response and provide protection from tick-borne spirochetes. VRPs expressing ospA that accumulated intracellularly (VRP OspA) or that was secreted from host cells (VRP tPA-OspA) were tested. Both VRP OspA and VRP tPA-OspA expressed ospA in immunized mice. Mice vaccinated with VRPs expressing secreted OspA produced significant amounts of anti-OspA antibodies, whereas VRPs expressing intracellular OspA were less immunogenic. The VRP method of delivery induced a Th1 type immune response unlike the recombinant OspA protein in Freund's adjuvant, which induced a mixed (Th1 and Th2) immune response. The VRP tPA-OspA construct induced an immune response that reduced the bacterial load in feeding Ixodes scapularis and blocked transmission to the host. These results indicate that VRPs are capable of providing protection against tick-borne B. burgdorferi, and potentially can be used for developing improved vaccines against Lyme disease.     

Head-to-head comparison on the immunogenicity of two HIV/AIDS vaccine candidates based on the attenuated poxvirus strains MVA and NYVAC co-expressing in a single locus the HIV-1BX08 gp120 and HIV-1(IIIB) Gag-Pol-Nef proteins of clade B

In this investigation we have generated and defined the immunogenicity of two novel HIV/AIDS vaccine candidates based on the highly attenuated vaccinia virus strains, MVA and NYVAC, efficiently expressing in the same locus (TK) and under the same viral promoter the codon optimized HIV-1 genes encoding gp120 and Gag-Pol-Nef antigens of clade B (referred as MVA-B and NYVAC-B). In infected human HeLa cells, gp120 is released from cells and GPN is produced as a polyprotein; NYVAC-B induces severe apoptosis but not MVA-B. The two poxvirus vectors showed genetic stability of the inserts. In BALB/c and in transgenic HHD mice for human HLA-A2 class I, both vectors are efficient immunogens and induced broad cellular immune responses against peptides represented in the four HIV-1 antigens. Some differences were observed in the magnitude and breadth of the immune response in the mouse models. In DNA prime/poxvirus boost protocols, the strongest immune response, as measured by fresh IFN-gamma and IL-2 ELISPOT, was obtained in BALB/c mice boosted with NYVAC-B, while in HHD mice there were no differences between the poxvirus vectors. When the prime/boost was performed with homologous or with combination of poxvirus vectors, the protocols MVA-B/MVA-B and NYVAC-B/NYVAC-B, or the combination NYVAC-B/MVA-B gave the most consistent broader immune response in both mouse models, although the magnitude of the overall response was higher for the DNA-B/poxvirus-B regime. All of the immunization protocols induced some humoral response against the gp160 protein from HIV-1 clone LAV. Our findings indicate that MVA-B and NYVAC-B meet the criteria to be potentially useful vaccine candidates against HIV/AIDS.     

Induction of gp120-specific protective immune responses by genetic vaccination with linear polyethylenimine-plasmid complex

The induction of IFN-gamma-secreting CD8+ T cells and neutralizing antibodies to HIV-1 are both key requirements for prevention of viral transmission and clearance of pathogenic HIV. Although DNA vaccination has been shown to induce both humoral and cellular immune responses against HIV antigens, the magnitude of the immune responses has always been disappointing. In this report, we analyze the ability of polyethylenimine (PEI)-DNA complex expressing an HIV-glycoprotein 120 (gp120) antigen (PEI-pgp120) to induce systemic CD8+ T cell and humoral responses to the gp120 antigen. The administration of PEI-plasmid complex resulted in rapid elevation of serum levels of IL-12 and IFN-gamma. Furthermore, a single administration of PEI-pgp120 complex elicits a number of gp120-specific CD8+ T cells 20 times higher than that elicited by three intramuscular injections of naked DNA. Interestingly, we found that systemic vaccination with PEI-pgp120 induced protective immune responses against both systemic and mucosal challenges with a recombinant vaccinia virus expressing a gp120 antigen. The data also demonstrated that the depletion of macrophages with liposome-encapsulated clodronate completely abolished gp120-specific cellular response. Overall, our results showed that a single administration of PEI-pgp120 complexes, eliciting strong immune responses, is an effective vaccination approach to generate protection against systemic and mucosal viral infections.     

Ginseng stem-leaf saponins (GSLS) and mineral oil act synergistically to enhance the immune responses to vaccination against foot-and-mouth disease in mice

Saponins extracted from ginseng stems and leaves (GSLS) as well as the synergistic effect between GSLS and oil emulsion were investigated for their adjuvant effects on the immune responses of mice to vaccination against foot-and-mouth disease virus (FMDV) serotype Asia 1. In experiment A, ICR mice were subcutaneously immunized twice with FMDV antigen with or without GSLS (0, 1, 5, 10 and 20 microg) at 3 week intervals. Highest FMDV-specific IgG level was observed 2 weeks after the boosting in mice immunized with FMDV antigen plus 10 microg of GSLS. In experiment B, mice were subcutaneously injected with FMDV antigen with or without GSLS (10 microg), or in oil emulsion with or without GSLS (10 microg) on days 1 and 21. Results indicated that when co-administered with a mixture of oil and GSLS, FMDV antigen induced significantly higher IgG titer and IgG1, IgG2a, IgG2b and IgG3 responses, production of IFN-gamma (Th1 cytokine) and IL-5 (Th2 cytokine) by splenocytes, as well as T and B lymphocyte proliferation in response to Con A and LPS than when FMDV antigen was used alone or mixed with either GSLS or oil. This suggests that GSLS and oil adjuvant synergistically promote both Th1 and Th2 immune responses. As protection against FMDV requires both cellular and humoral immune responses, the combined effects of GSLS and oil deserve further study in other animals such as cattle and pigs in order to induce effective immunity against FMDV infection.     

Vaccination with coxsackievirus B3 virus-like particles elicits humoral immune response and protects mice against myocarditis

Coxsackievirus B3 (CVB3), along with other enteroviruses, is involved in about 50% of myocarditis cases and in the pathogenesis of dilated cardiomyopathy. Prevention of CVB3 infection is therefore highly desirable. Virus-like particles (VLPs) are structurally similar to native virus particles and therefore are far better immunogens than any other subunit vaccines. Recombinant baculoviruses carrying either the intact, entire coding region of CVB3 or the four individual coding regions for virus proteins 1-4 (VP1-4) were constructed. Expression of CVB3 capsid proteins in insect cells infected with recombinant baculovirus was detected by immunofluorescence and Western blot analysis. Sucrose gradient ultracentrifugation fractions of the infected cell lysates contained peaks of CVB3 antigen with an approximate density of 1.14g/ml. Electron microscopy demonstrated the presence of VLP in these sucrose fractions. The CVB3 VLP was non-infectious in tissue culture. SWR (H-2(q)) mice vaccinated with CVB3 VLP developed antibodies to CVB3 capsid proteins after the first boost. Antibody titre was comparable to the level induced by an attenuated CVB3 vaccine. Vaccinated animals were protected from myocarditis when subsequently challenged with cardiovirulent CVB3 (chimera-2). Vaccination with VLP produced from the complete CVB3 coding region gave a greater immune response and afforded better protection than with VLP from the quadruple expression vector. These results demonstrate that CVB3 capsid proteins expressed in insect cells have the intrinsic capacity to assemble into non-infectious VLP, which afforded protection from CVB3 infection to mice when used as a vaccine.     

Baculovirus vectors expressing F proteins in combination with virus-induced signaling adaptor (VISA) molecules confer protection against respiratory syncytial virus infection

Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV.     

Protective immunity against foot-and-mouth disease virus induced by a recombinant vaccinia virus

We report the construction of a recombinant vaccinia virus expressing the precursor for the four structural proteins of FMD virus (FMDV) (P1) strain C3Arg85 using a procedure for isolation of recombinant vaccinia viruses based solely on plaque formation. Adult mice vaccinated with this recombinant vaccinia virus elicited high titers of neutralizing antibodies against both the homologous FMDV and vaccinia virus, measured by neutralization assays. Liquid phase blocking sandwich enzyme-linked immunosorbent assays (ELISAs) using whole virus as antigen showed high total antibody titers against homologous FMDV, similar to those induced by the conventional inactivated vaccine. When ELISAs were carried out with heterologous strains A79 or O1Caseros as antigens, sera from animals vaccinated with the recombinant virus cross-reacted. Mice boosted once with the recombinant vaccinia virus were protected against challenge with infectious homologous virus. These results indicate that recombinant vaccinia viruses are efficient immunogens against FMDV when used as a live vaccine in a mouse model.     

Induction of protective immunity in swine by immunization with live attenuated recombinant pseudorabies virus expressing the capsid precursor encoding regions of foot-and-mouth disease virus

Foot-and-mouth disease (FMD) causes morbidity to livestock and serious economic consequences to its associated industry and therefore it is necessary to develop a safe and efficient vaccine to prevent or control this disease. A recombinant live attenuated virus vaccine, designated PRV-P1, was generated by insertion of an expression cassette containing CMV promoter, FMDV P1 gene and SV 40 poly-A into the gG gene region of a live attenuated pseudorabies virus vaccine strain (TK⁻/gG⁻/LacZ⁺). To determine the induction of protective immunity, 16 FMDV and PRV seronegative white swine were randomly divided into four groups and immunized intramuscularly. The parental virus (TK⁻/gG⁻/LacZ⁺) was injected into three pigs, the recombinant virus PRV-P1 into five pigs and commercial FMD-inactivated vaccine into five pigs, with PBS (negative control) into three pigs. All animals were immunized again 4 weeks later to boost the immune response and challenged with virulent type O FMDV O/ES/2001 strain 4 weeks after the second immunization. Results showed PRV-P1 vaccinated pigs induced high-level neutralizing antibody response to both FMDV and PRV, and strong CTL response against FMD antigen activation. Three of five pigs were completely protected against challenge with FMDV, one pig minimally protected and the other one had increased protection but not complete. However, one pig vaccinated with commercial FMD vaccine developed constant pyrexia. Average levels of antibodies against non-structural 3ABC proteins were significantly lower and efficacy on inhibition of FMDV replication was much increased in swine vaccinated with PRV-P1 than those immunized with commercial FMD vaccine after FMDV challenge. Our results showed that the recombinant PRV-P1 can induce not only humoral and cell-mediated immune responses but also partial protection against FMDV challenge, making it a good candidate for future development of the FMD vaccine.     

Heterotypic protection from rotavirus infection in mice vaccinated with virus-like particles

Virus-like particles (VLPs) composed of rotavirus VP2, VP6, and VP7 of G1 or G3 serotype specificity were produced in insect cells coinfected with recombinant baculoviruses expressing single rotavirus genes. The VLPs were purified and subsequently evaluated for immunogenicity and protection in the adult mouse model of rotavirus infection. Mice were vaccinated twice intramuscularly with G1 VLPs formulated with Quillaja saponaria (QS-21) or adsorbed to aluminium hydroxide (AlOH), or with G1 VLPs alone. G3 VLPs, G1 plus G3 VLPs, inactivated SA11 virions formulated with QS-21, or adjuvants were similarly inoculated as controls. Mice were examined for serum and fecal antibody responses by ELISA or microneutralization assays. Protective efficacy of the VLP vaccine formulations against oral challenge with the G3 murine ECwt rotavirus was assessed by comparing the antigen shed in stool of the VLP-vaccinated mice to that of the adjuvant-immunized mice. G1 VLPs in QS-21 induced significantly higher serum and intestinal antibody titers than G1 VLPs in AlOH or G1 VLPs alone. QS-21 also heightened serum and fecal antibody responses to G3 VLPs. These QS-21-augmented antibody responses were further characterized by equivalent IgG1 and IgG2a titers in sera, suggesting that G1 or G3 VLPs in QS-21 induced a balanced Th1/Th2 response. G1 VLPs in QS-21 induced partial protection (88%) against oral challenge with the heterotypic ECwt virus, whereas G3 VLPs in QS-21 induced complete protection (100%). In contrast, G1 VLPs when formulated with AlOH induced a predominant Th2 response and did not protect (1%) mice from virus challenge. Our results indicate that the type of adjuvant used clearly influences both antibody responses to rotavirus VLPs and the protective efficacy against rotavirus infections. These data have important implications for the development of parenteral vaccines to ameliorate rotavirus disease.     

The HIV-1 chimeric protein CR3 expressed by poxviral vectors induces a diverse CD8+ T cell response in mice and is antigenic for PBMCs from HIV+ patients

Recombinant avipoxvirus vectors are attractive for vaccination against human immunodeficiency virus type 1 (HIV-1), where induction of a cytotoxic CD8(+) T cell (CTL) response seems to be an important component of protective immunity. We expressed the chimeric protein CR3, composed by CTL epitopes rich regions from, RT, Gag and Nef and conserved Th cell epitopes from gp120, gp41 and Vpr of HIV-1 in a fowlpox virus (FWPV) vector (FPCR3), and used this vector to induce HIV-specific CTL responses in mice. Mice immunised twice intraperitoneally with FPCR3, developed a CD8(+) T cell response measured as production of IFN-gamma by splenocytes in response to stimulation with P815 cells infected with recombinant vaccinia viruses (rVV) expressing CR3, Gag and Nef. The number of IFN-gamma secreting cells was markedly higher when a P815 cell line constitutively expressing CR3 was used as target cells for Enzyme-linked-immunospot (ELISPOT). CR3 epitopes were also specifically recognised by human PBMCs from three HIV(+) patients with different haplotypes. These results confirm the potential of FWPV vectors expressing these novel HIV-1 chimeric proteins to induce a simultaneous CD8(+) T cell response against conserved viral targets and early expressed regulatory proteins.     

水痘-带状疱疹病毒糖蛋白I基因在昆虫细胞中的表达及纯化

目的 将北京水痘-带状疱疹病毒(VZV)84-7株克隆糖蛋白I(gpI)基因在杆状病毒-昆虫细胞表达系统中表达,并对其表达产物进行纯化。方法 采用PCR方法从VZVDNA中扩增gpI全基因序列,并将其插入杆状病毒转移质粒pBacPAK9中,获得重组转移质粒pBacVZVgpI,对pBacVZVgpI中的插入基因进行测序。重组转移质粒与线性杆状病毒BacPAK6DNA(Bsu36Idigested)共转染Sf9昆虫细胞,获得重组病毒BacPAK-gpI。通过亲和层析纯化重组蛋白,并检测其抗原性。结果 PCR扩增得到gpI基因,测序结果表明克隆的外源基因正确。经SDS-聚丙烯酰胺凝胶电泳(SDS—PAGE)、免疫印迹(weotem-blot)方法证明gpI基因在昆虫细胞中获得表达,表达产物在培养72h达到高峰,重组蛋白的相对分子质量约为58000和70000,与理论值相符,蛋白质加工与天然蛋白类似。动物实验结果表明,重组蛋白具有较好的免疫原性,可刺激小鼠产生中和抗体。SDS—PAGE检测纯化的重组蛋白,纯度达80％。纯化蛋白经western-blot和ELISA检测后显示,具有特异的抗体结合活性。结论 应用昆虫细胞表达水痘-带状疱疹病毒gpI基因,可为水痘-带状疱疹病毒抗原定量分析、糖蛋白ELISA的研制和制备亚单位疫苗提供基础。     

Vaccination with an adenoviral vector encoding hepatitis C virus (HCV) NS3 protein protects against infection with HCV-recombinant vaccinia virus

Cellular immune response plays an important role in the clearance of hepatitis C virus (HCV). Thus, development of efficient ways to induce anti-viral cellular immune responses is an important step toward prevention and/or treatment of HCV infection. With this aim, we have constructed a replication-deficient recombinant adenovirus expressing HCV NS3 protein (RAdNS3). The efficacy of RAdNS3 was tested in vivo by measuring the protection against infection with a recombinant vaccinia virus expressing HCV-polyprotein (vHCV1-3011). Immunisation with 10(9)pfu of RAdNS3 induced anti-NS3 humoral, T helper and T cytotoxic responses. We identified eight epitopes recognised by IFN-gamma producing cells, five of them exhibiting lytic activity. Moreover, we show that RAdNS3 immunised mice were protected against challenge with vHCV1-3011 and that this protection was mediated by CD8(+) cells. In conclusion, our results suggest that adenoviral vectors encoding NS3 might be useful for the induction of prophylactic and/or therapeutic anti-HCV immunity.