Staphylococcus aureus capsular polysaccharide type 5 conjugate and whole cell vaccines stimulate antibody responses in cattle.

Dairy heifers were immunized subcutaneously with one of four different vaccines which contained preparations of Staphylococcus aureus capsular polysaccharide type 5 (CP5) and a mineral oil adjuvant, or received a placebo containing saline and adjuvant. The vaccine containing a CP5-human serum albumin conjugate (CP5-HSA) and the vaccine with formaldehyde inactivated whole cells expressing CP5, both elicited strong anti-CP5 antibody responses. After two injections three weeks apart and a third injection 10 months later, the mean level and duration of the anti-CP5 antibody response was significantly higher in the whole cell group. No differences were found between the two groups with regard to the relative proportion of IgG subclasses, and the antibody responses to the polysaccharide were composed of both the IgG1 and IgG2. Vaccines containing only free CP5 or CP5 mixed with HSA produced weak and transient humoral immune responses. Only animals vaccinated with the whole cell vaccine or the conjugate vaccine showed responses to CP5 in a lymphocyte proliferation assay conducted one year after the third vaccination. This study indicates that CP5 expressed on the surface of formaldehyde inactivated whole cells, emulsified in an oil adjuvant, gives a strong and long lasting immune response in cattle. The use of conjugation technology, although effective, might not be necessary in order to achieve an immune response against S. aureus CP5 in cattle.     

Immunogenicity of liposomal malaria sporozoite antigen in monkeys: adjuvant effects of aluminium hydroxide and non-pyrogenic liposomal lipid A

The immunogenicity of a recombinant protein (R32tet₃₂) containing sequences from the tetrapeptide repeat region of the circumsporozoite protein of Plasmodium falciparum was enhanced by encapsulation in liposomes containing lipid A and adsorption of the liposomes with alum. The toxicities and efficacies of preparations containing different types and doses of lipid A were assessed by studying pyrogenicity in rabbits and adjuvanticity in monkeys. In each case liposomal lipid A was 25-fold to 200-fold less pyrogenic than free lipid A. Monophosphoryl lipid A, whether free or in liposomes, was the least pyrogenic of the three lipid A preparations tested. High antibody levels were obtained after immunization of rhesus monkeys with a formulation consisting of alum-adsorbed liposomes in which the liposomes contained R32tet₃₂ and a strongly pyrogenic dose of native lipid A. Excellent antibody levels were also observed in monkeys immunized with a combination of R32tet₃₂ encapsulated in alum-adsorbed liposomes containing non-pyrogenic doses of monophosphoryl lipid A and alum. The adjuvant effect was related to the dose of the lipid A in the liposomes, and the adjuvant effect was still strongly expressed despite suppression of the pyrogenic effect of lipid A. Antibody levels were considerably lower in monkeys immunized with liposomes lacking lipid A. It was concluded that a non-pyrogenic formulation of alum-adsorbed liposomes, in which the liposomes contained both lipid A and an encapsulated synthetic sporozoite antigen, shows considerable promise for inducing high titres of antibodies to sporozoites.     

Development of a new type of influenza subunit vaccine made by muramyldipeptide-liposome: enhancement of humoral and cellular immune responses

The muramyldipeptide (MDP), [6-O-(2-tetradecyl-hexa-decanoyl)-N-acetylmuramyl-L-isoglutamine] can be incorporated into liposomes with haemagglutinin and neuraminidase subunits were attached to the inner and outer surfaces of lamellar structures of the liposomes, probably through their hydrophobic ends. The addition of cholesterol resulted in much more stable liposomes, which were similar in size and shape to native influenza virus particles. These liposomes enhanced the immunogenicity of haemagglutinin in mice, such that the levels of antibody induced were about 16-fold higher than those of subunit haemagglutinin vaccine alone. Results of proliferation tests with spleen cells from mice and guinea-pigs were consistent with the immunopotentiation of haemagglutinin by liposomes. In addition, the higher antibody levels produced in mice, immunized with the haemagglutinin and MDP-containing liposomes (MDP-virosomes), were maintained for at least 6 months. Enhancement of the cellular immune response, measured by delayed type hypersensitivity reactions, was also observed in the guinea-pigs immunized with MDP-virosome vaccine. Preliminary tests with splenocytes from mice immunized with different vaccines also indicated that the MDP-virosome vaccine induced cytotoxic T-cell activity in these mice. This study revealed that the formation of liposomes with muramyldipeptide enhanced the level and persistence of circulating antibody, and enhanced cellular immunity in guinea-pigs and mice.     

Immune response and protection assay of recombinant major surface glycoprotein of Leishmania (rgp63) reconstituted with liposomes in BALB/c mice

In this study the ability of recombinant gp63 entrapped in liposomes to induce immune response and protection against L. major infection in susceptible BALB/c mice was studied. Liposomes containing rgp63 (Lip-rgp63) were prepared from egg lecithin and cholesterol using detergent solubilization method. Immunization of BALB/c mice with rgp63 alone conferred a partial protection while entrapment of rgp63 in liposomes significantly increased the rate of protection (P<0.05). The parasite burden of spleen in mice challenged with L. major was significantly (p<0.001) lower in group of mice immunized with rgp63 alone or Lip-rgp63, however, the least parasite burden was seen in Lip-rgp63 group. Both rgp63 alone and Lip-rgp63 elicited significant delayed-type hypersensitivity (DTH) response compared to controls (p<0.01), however, the DTH response of PBS-rgp63 was less than the Lip-rgp63. Titration of anti-Leishmania IgG isotypes (IgG2a/IgG1) showed a preferential Th1 type of immune response only in mice immunized with Lip-rgp63. The results indicate that liposomes might be used as a suitable immunoadjuvant for development of Leishmania vaccine.     

Humoral immune response elicited in rats by measles viral membrane antigens presented in liposomes and ISCOMs

The immunogenicity of measles virus glycoproteins presented associated to liposomes or ISCOMs was compared with that of whole virus and solubilized membrane proteins in W/Fu rats. The rats were immunized three times at ten-day intervals with syngeneic peritoneal exudate cells fed in vitro with the various antigen preparations. A strong and persisting antibody response with haemagglutinin inhibitory and neutralization activity was observed in rats immunized with liposomes, ISCOMs, or virus. The responses were very similar despite the lower dose of protein received by rats immunized with ISCOMs (1 microgram protein) or with liposomes (20 micrograms protein). By contrast, injection of peritoneal exudate cells previously fed in vitro with soluble H and F glycoproteins resulted in only a poor and transient response. The sera from rats immunized with virus, liposomes or ISCOMs contained antibodies immunoprecipitating mainly H and F glycoproteins. Despite a strong enrichment in F polypeptides during the preparation of ISCOMs, they induced an equal anti-H and anti-F antibody response.     

Enhanced protection against pneumococcal infection elicited by immunization with the combination of PspA, PspC, and ClpP

Immunization with a combination of several virulence-associated proteins is one of the strategies of developing effective protein-based vaccines to enhance the protection against Streptococcus pneumoniae. In this study, we evaluated the protection effects against pneumococcal infection caused by S. pneumoniae TIGR4 in BALB/c mice immunized with either single pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), the caseinolytic protease (ClpP) or their combinations. The median survival times for mice immunized with single antigen or their combinations were significantly longer than that for mice treated with adjuvant alone. Mice treated with a combination of three antigens survived significantly longer than those that received either single or two antigens. The highest survival rate of the various groups of mice was observed with the combination of three antigens, this survival rate was significantly different from those for mice that received either single antigen or the combinations of two antigens except the mixture of ClpP and PspA. In the experiment of passive immunization with hyperimmune serums containing their specific polyclonal antibodies (anti-PspA serum, anti-PspC serum, anti-ClpP serum), the median survival times for mice immunized with hyperimmune serums containing specific polyclonal antibodies were significantly longer than that for control mice, the treatment of serum containing only one single polyclonal antibody could not provide higher survival rate than control serum. However, the survival rates for mice treated with the serums containing combined polyclonal antibodies were significantly higher than those for mice treated with either control serum or anti-PspA serum alone. Immunization with the combination of three hyperimmune serums also provided the best protection against S. pneumoniae. Compared to mice treated with serum containing single polyclonal antibody, the survival rate for mice treated with serums containing three polyclonal antibodies was significantly higher but was not different from those for mice treated with serums containing two polyclonal antibodies. Our findings provided evidence that a mixture of PspA, PspC, and ClpP or their polyclonal antibodies could enhance the protection against pneumococcal infection acting a synergetic effect.     

Immunogenicity in mice of pneumococcal glycoconjugate vaccines using pneumococcal protein carriers

Antibody response and protective immunity were evaluated in mice immunized with pneumococcal glycoconjugate vaccines using two pneumococcal protein carriers. Mice injected with type 9V polysaccharide (PS) conjugated to inactivated pneumolysin (Ply) or autolysin (Aly) produced high levels of IgG and IgM antibodies to both the PS and the protein carrier. Higher PS antibody titers to the pneumococcal PS conjugates were measured by ELISA using PS-Ply or PS-tetanus toxoid (TT) conjugate as a coating antigen compared with PS mixed with methylated human serum albumin. Type 9V PS (10 microg) inhibited most of the 9V IgM and IgG antibody binding to the 9V-TT coated plate. In contrast, absorption with 19F PS did not inhibit 9V antibody binding. The avidity index of IgG antibodies in the 9V PS-Ply serum was 55.5 +/- 0.9, compared with 47.8 +/- 1.4 for 9V PS-Aly serum. Thus, high avidity of serum antibodies in conjugate-immunized mice can provide more effective functional activity for protection against pneumococcal infection. Mice immunized with these glycoconjugates exhibited rapid bacterial clearance from blood and provided cross-protection against challenge with heterologous serotypes of virulent pneumococci. These results reveal that conjugates using pneumococcal protein carriers can induce opsonophagocytic activity to destroy homologous and heterologous pneumococci, indicating that such conjugates can confer broader protective immunity than conjugates using non-pneumococcal proteins.     

Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253-275) and nucleoprotein (N protein) (amino acids 404-418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.     

DNA vaccination against respiratory influenza virus infection

DNA vaccination using plasmid encoding the hemagglutinin (HA) gene of influenza A/PR/8/34 virus to induce long-lasting protective immunity against respiratory infection was evaluated in this study. Using liposomes as carriers, the efficacy of DNA vaccines was determined using a lethal influenza infection model in mice. Mice immunized intranasally or intramuscularly with liposome-encapsulated pCI plasmid encoding HA (pCI-HA10) were completely protected against an intranasal 5 LD(50) influenza virus challenge. Mice immunized with liposome-encapsulated pCI-HA10, but not naked pCI-HA10, by intranasal administration were found to produce high titers of serum IgA. These results suggest DNA vaccines encapsulated in liposomes are efficacious in inducing complete protective immunity against respiratory influenza virus infection.     

Effect of immunological adjuvant combinations on the antibody and T-cell response to vaccination with MUC1-KLH and GD3-KLH conjugates

A year ago we described a comparison of 19 immunological adjuvants for their ability to augment antibody and T-cell responses against vaccines containing two cancer antigens, GD3 ganglioside and MUC1 peptide, covalently attached to keyhole limpet hemocyanin (KLH). As in our previous experience, the saponin fraction QS-21 was the most potent single adjuvant but several other adjuvants also had potent adjuvant activity. Induction of an immune response against cancer antigens is generally difficult because these antigens are autoantigens. To get maximal benefit from the adjuvant component of cancer vaccines we have now tested whether combinations of the optimal adjuvants induced an improved immune response compared to QS-21 alone. Since over the intervening year a new semi-synthetic saponin adjuvant (GPI-0100) containing the dodecylamide derivative of hydrolyzed naturally-occurring saponins had become available, this was tested as well. Twelve different adjuvant combinations and GPI-0100 were compared for their ability to augment (1) antibody responses against GD3 and MUC1 and (2) T-cell responses against GD3, MUC1 and KLH. GPI-0100 and five adjuvant combinations were superior to QS-21 alone for induction of IgM and IgG antibodies against MUC1 and/or GD3: QS-21 plus bacterial nucleotide CpG, QS-21 plus monophosphoryl lipid A (MPL), QS-21 plus non-ionic block copolymer CRL-1005, QS-21 plus Titermax and Titermax plus CpG. Antibody responses were documented both by ELISA against purified antigens and by FACS for cell surface reactivity. There was no evidence for T-cell immunity against GD3 or MUC1. The antibody responses against GD3 and MUC1 were, however, strongly correlated with IFN-gamma release and DTH against KLH. These results demonstrate that combinations of immunological adjuvants are able to augment antibody and T-cell responses to these conjugates beyond that attainable with QS-21 alone, and again confirm the absolute necessity of potent adjuvants or adjuvant combinations for optimal immunogenicity with conjugate vaccines.     

Immunological properties of a DNA plasmid encoding a chimeric protein of herpes simplex virus type 2 glycoprotein B and glycoprotein D

A DNA plasmid containing a chimeric sequence encoding both herpes simplex virus type 2 (HSV-2) glycoprotein B (gB) and glycoprotein D (gD) external domains (pcgDB) was used to immunize BALB/c mice against genital HSV-2 infection. To determine the efficacy of this vaccine, groups of mice immunized with the pcgDB plasmid were compared with animals immunized with plasmids corresponding to the individual proteins (pcgBt or pcgDt), administered separately or in combination (pcgBt + pcgDt). We studied the response of the different mouse groups to viral challenge by analyzing clinical disease (vaginitis), serum antibody levels, as well as lymphoproliferative responses and cytokine production by spleen cells. Increased IFN-gamma levels correlated with prolonged survival in mice immunized with the plasmid pcgDB, relative to mice immunized with plasmids coding for the individual proteins alone or in combination. Our results show that immunization with the plasmid encoding the chimeric protein is advantageous over separate proteins. These findings may have important implications for the development of multivalent DNA vaccines against HSV and other complex pathogens.     

Non-coding pDNA bearing immunostimulatory sequences co-entrapped with leishmanial antigens in cationic liposomes elicits almost complete protection against experimental visceral leishmaniasis in BALB/c mice

The difficulty in making successful vaccines against leishmaniasis is partly due to lack of an appropriate adjuvant. Non-coding plasmid DNA (pDNA) bearing immunostimulatory sequences (ISS) is a potent activator of innate immunity, and can thus act as an adjuvant with vaccine antigen. We therefore evaluated the efficacy of pDNA and soluble leishmanial antigens (SLA) to protect against challenge with Leishmania donovani infection. We demonstrate that immunomodulatory activity of pDNA, which potentiated a Th1 immune responses, led to enhanced protection with SLA. Importantly, adding cationic liposomes as vehicle to the antigen, with pDNA either complexed or entrapped within, significantly increased the potentiating effect of pDNA. Further, comparison of the two vaccine formulations demonstrated an impressive increase in the protective efficacy up to two folds when both antigen and pDNA were within the vehicle. Thus, these studies establish the utility of non-coding pDNA bearing ISS as strong promoters of vaccine potency of liposomal antigens especially when co-entrapped with the antigen in cationic liposomes.     

Plasma beads loaded with Candida albicans cytosolic proteins impart protection against the fungal infection in BALB/c mice

The development of a prophylactic vaccine against systemic candidiasis, employing Candida albicans cytosolic proteins (Cp) as antigen and fibrin cross-linked plasma beads as an antigen bearing dual delivery system is described. Groups of mice were administered either with free Cp, or Cp entrapped in plasma beads, Cp entrapped in liposomes or liposome encapsulated Cp further entrapped in plasma beads. Humoral immunity was studied by measuring the anti-Cp antibody titers in the sera of the immunized animals. Induction of cell-mediated immunity was assessed by delayed type hypersensitivity (DTH), NO production, up-regulation of co-stimulatory molecules viz. CD80, CD86 on APCs on one hand and T-cells proliferation as well as induction of IFN-γ and IL-4 on the other. The efficacy of various vaccine formulations in protecting mice against a lethal challenge with C. albicans, was assessed by determining animal survival rate and fungal burden in the systemic circulation and vital organs. Among various Cp-based vaccines investigated, the preparation containing liposomized Cp entrapped in plasma beads imparted superior protection in the immunized mice as compared to other antigens delivery systems.     

抗桔青霉素单克隆抗体的研制与鉴定

目的制备抗桔青霉素的单克隆抗体。方法采用活性酯法、甲醛加成法和羰基二咪唑法制备了4种桔青霉素与载体蛋白的偶联物(A、B、C和D),免疫小鼠制备抗桔青霉素单克隆抗体。结果免疫原性鉴定证明偶联物C可刺激BALB/c小鼠产生抗桔青霉素的抗体,细胞融合后筛选到一株抗桔青霉素的单克隆抗体,单抗与赭曲霉毒素A、黄曲霉毒素B1和展青霉素等毒素交叉反应低于0.01%,在此基础上建立了竞争ELISA检测方法,线性范围为20~10,00ng/ml,检测下限为10ng/ml。在小麦样品中的加标回收率为95%~112%,变异系数为9.1%~18.6%。结论本研究成功筛选到抗桔青霉素单克隆抗体。     

DNA vaccines co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV) display enhanced immunogenicity

The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In the present study, three different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells and heterodimerization altered the subcellular localization of GP5. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 6 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibody could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. These results indicate that the formation of GP5/M heterodimers may be involved in post-translational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.     

Oligonucleotide containing CpG motifs enhances immune response to mucosally or systemically administered tetanus toxoid

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides induce proliferation of B cells and activation of macrophages and thus stimulation of the immune system. We tested an oligonucleotide containing an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines (GAGAACGCTCGACCTTCGAT) for the ability to affect antibody levels to tetanus toxoid (Tt). Groups of male Rowett rats (n=5-6/group) received colloidal aluminium hydroxide (Al(OH)3) either alone, or with Tt bound to the Al(OH)3, or with Tt bound to Al(OH)3 with the addition of the CpG oligonucleotide. Antigens were administered subcutaneously in the salivary gland vicinity once, or by gastric intubation on 3 consecutive days. On day 124 all animals were given a boost with the same material by the same route. Serum IgG and saliva IgA antibody to Tt was determined by ELISA. Serum antibody levels were significantly higher in ODN+Tt treated rats than in Tt-alone rats immunized by either route after primary or booster immunizations. Thus, administration of an ODN containing unmethylated CpG motifs along with an immunogen bound to Al(OH)3 can result in enhanced specific antibody when administered by intragastric as well as subcutaneous routes.     

Enhanced immune protection by a liposome-encapsulated recombinant respiratory syncytial virus (RSV) vaccine using immunogenic lipids from Deinococcus radiodurans

The radiation-resistant bacterium, Deinococcus radiodurans contains a variety of phospho-, glyco- and phosphoglycolipids, the structures of which appear to be largely unique in nature. We show here that such lipids are immunogenic when administered as liposomes intranasally in mice, as evidenced by the induction of serum antibodies which recognize D. radiodurans lipids but not other lipids by thin layer chromatographic immunostaining. By modifying a liposomal vaccine against respiratory syncytial virus (RSV) we find that vaccine efficacy is markedly enhanced by the inclusion of lipids isolated from D. radiodurans. Using dioleoylphosphatidylcholine (DOPC) or D. radiodurans lipids, liposomes were prepared which encapsulated a soluble fragment of the RSV G protein (G(128-188)) fused with a portion of the bacterial thioredoxin (Trx) protein. Mice immunized intranasally with D. radiodurans liposomes showed markedly greater protection against RSV challenge over mice immunized with DOPC liposomes. Enhanced vaccine efficacy was achieved using liposomes prepared from either whole D. radiodurans lipids or from a single isolated phosphoglycolipid previously identified as alpha-galactosylphosphatidylglyceroylalkylamine (lipid 7). Mice immunized and protected against RSV challenge were free of pulmonary eosinophilic infiltration, an undesirable consequence of many RSV vaccines. The results provide further support for liposome-based vaccines for RSV and underline the importance of lipid composition in liposome formulations.     

Intranasal immunization with liposome-formulated Yersinia pestis vaccine enhances mucosal immune responses

The induction of mucosal immune responses by a liposome-formulated Y. pestis vaccine (formaldehyde-killed whole cell vaccine; KWC) was evaluated. We demonstrated that intranasal immunization of mice with Y. pestis KWC vaccine, formulated with liposomes, significantly enhanced mucosal immune responses in the lung when compared to the responses induced with KWC vaccine alone. These immune responses were characterized by increased titres of specific IgA and IgG in mucosal secretions (lung and nasal washes), and an increased frequency of specific antibody-secreting cells in the lungs. In addition, antigen-specific proliferative responses and IFN-gamma-secreting cells were also significantly enhanced in the spleens of mice immunized with the KWC vaccine formulated in liposomes. Animals that were immunized intranasally with the KWC vaccine showed significant protection against an intranasal challenge with Y. pestis. These results highlight the importance of mucosal administration of vaccine antigens to stimulate immunity in the respiratory tract and demonstrate that liposome formulations can improve the effectiveness of conventional vaccines.     

Protective immune response in mice vaccinated with a recombinant adenovirus containing capsid precursor polypeptide P1, nonstructural protein 2A and 3C protease genes (P12A3C) of encephalomyocarditis virus

Encephalomyocarditis virus (EMCV) infection can cause acute myocarditis and sudden death in pre-weaned piglets as well as severe reproductive failure in sows. In this study, two recombinant adenoviruses containing capsid precursor polypeptide P1 alone (Ad-P1) and P1 plus nonstructural protein 2A and 3C protease coding regions (Ad-P12A3C) of EMCV were respectively constructed using replication-defective human adenovirus serotype 5 as vector, and their antibody responses and protective efficacies against a lethal EMCV challenge were evaluated in mice. Both Ad-P1 and Ad-P12A3C were confirmed to be capable of expressing VP1 protein in BHK21 cells by immunoperoxidase monolayer assay (IPMA). The results showed that mice vaccinated once or twice with Ad-P1 and Ad-P12A3C generated specific antibody response against VP1 protein of EMCV. Although Ad-P1 induced higher antibody titers, virus-neutralizing antibody response was considerably less (p<0.05), compared to that of Ad-P12A3C. Upon challenging with a virulent EMCV strain, Ad-P12A3C elicited efficacious protection (100% for both vaccination once and twice) in the vaccinated mice; whereas the mice immunized with Ad-P1 showed a lower protection (12.5% for vaccination once and 75% for twice). Our work suggests that the recombinant adenovirus (Ad-P12A3C) containing the capsid precursor polypeptide coding region (P1) plus nonstructural protein 2A and 3C protease genes have an excellent potential to be used as a vaccine that can provide sufficient protective efficacy against EMCV infection in animals.     

Ability of synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa to afford protection against P. aeruginosa infection in a murine acute pneumonia model

Three synthetic peptides (Nos 9, 10 and 18) representing surface-exposed, linear B-cell epitopes of outer membrane protein F of Pseudomonas aeruginosa were each conjugated to the carriers keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA), with the conjugates being used to immunize mice intranasally. Mice were also immunized intranasally with a KLH/BSA carrier control or with a peptide No. 8 conjugate as a negative control. An immunoglobulin G response reactive with P. aeruginosa whole cells was demonstrated by enzyme-linked immunosorbent assay (ELISA) of sera from mice immunized with peptide 9, 10 or 18, whereas no whole-cell reactivity by ELISA was detected in sera from mice immunized with peptide 8. Upon pulmonary challenge of immunized mice with a Fisher-Devlin immunotype 4 strain of P. aeruginosa only those mice immunized with peptide 9 or peptide 10 had a significantly greater survival rate compared to control mice immunized with the carriers alone. Peptides 9 (TDAYNQKLSERRAN) and 10 (NATAEGRAINRRVE) have potential for further development as a protective vaccine against P. aeruginosa infections.