Potential use of a recombinant replication-defective adenovirus vector carrying the C-terminal portion of the P97 adhesin protein as a vaccine against Mycoplasma hyopneumoniae in swine

Mycoplasma hyopneumoniae causes severe economic losses to the swine industry worldwide and the prevention of its related disease, enzootic porcine pneumonia, remains a challenge. The P97 adhesin protein of M. hyopneumoniae should be a good candidate for the development of a subunit vaccine because antibodies produced against P97 could prevent the adhesion of the pathogen to the respiratory epithelial cells in vitro. In the present study, a P97 recombinant replication-defective adenovirus (rAdP97c) subunit vaccine efficiency was evaluated in pigs. The rAdP97c vaccine was found to induce both strong P97 specific humoral and cellular immune responses. The rAdP97c vaccinated pigs developed a lower amount of macroscopic lung lesions (18.5 ± 9.6%) compared to the unvaccinated and challenged animals (45.8 ± 11.5%). rAdP97c vaccine reduced significantly the severity of inflammatory response and the amount of M. hyopneumoniae in the respiratory tract. Furthermore, the average daily weight gain was slightly improved in the rAdP97c vaccinated pigs (0.672 ± 0.068 kg/day) compared to the unvaccinated and challenged animals (0.568 ± 0.104 kg/day). A bacterin-based commercial vaccine (Suvaxyn^® MH-one) was more efficient to induce a protective immune response than rAdP97c even if it did not evoke a P97 specific immune response. These results suggest that immunodominant antigens other than P97 adhesin are also important in the induction of a protective immune response and should be taken into account in the future development of M. hyopneumoniae subunit vaccines.     

Evaluation of recombinant Mycoplasma hyopneumoniae P97/P102 paralogs formulated with selected adjuvants as vaccines against mycoplasmal pneumonia in pigs

Pig responses to recombinant subunit vaccines containing fragments of eight multifunctional adhesins of the Mycoplasma hyopneumoniae (Mhp) P97/P102 paralog family formulated with Alhydrogel^® or Montanide™ Gel01 were compared with a commercial bacterin following experimental challenge. Pigs, vaccinated intramuscularly at 9, 12 and 15 weeks of age with either of the recombinant formulations (n = 10 per group) or Suvaxyn^® M. hyo (n = 12), were challenged with Mhp strain Hillcrest at 17 weeks of age. Unvaccinated, challenged pigs (n = 12) served as a control group. Coughing was assessed daily. Antigen-specific antibody responses were monitored by ELISA in serum and tracheobronchial lavage fluid (TBLF), while TBLF was also assayed for cytokine responses (ELISA) and bacterial load (qPCR). At slaughter, gross and histopathology of lungs were quantified and damage to epithelial cilia in the porcine trachea was evaluated by scanning electron microscopy. Suvaxyn^® M. hyo administration induced significant serological responses against Mhp strain 232 whole cell lysates (wcl) and recombinant antigen F3_P216, but not against the remaining vaccine subunit antigens. Alhydrogel^® and Montanide™ Gel01-adjuvanted antigen induced significant antigen-specific IgG responses, with the latter adjuvant eliciting comparable Mhp strain 232 wcl specific IgG responses to Suvaxyn^® M. hyo. No significant post-vaccination antigen-specific mucosal responses were detected with the recombinant vaccinates. Suvaxyn^® M. hyo was superior in reducing clinical signs, lung lesion severity and bacterial load but the recombinant formulations offered comparable protection against cilial damage. Lower IL-1β, TNF-α and IL-6 responses after challenge were associated with reduced lung lesion severity in Suvaxyn^® M. hyo vaccinates, while elevated pathology scores in recombinant vaccinates corresponded to cytokine levels that were similarly elevated as in unvaccinated pigs. This study highlights the need for continued research into protective antigens and vaccination strategies that will prevent Mhp colonisation and establishment of infection.     

A recombinant chimera composed of R1 repeat region of Mycoplasma hyopneumoniae P97 adhesin with Escherichia coli heat-labile enterotoxin B subunit elicits immune response in mice

Swine mycoplasmal pneumonia (SMP), caused by fastidious bacterium Mycoplasma hyopneumoniae, is the most important respiratory disease in swine breeding. The commonly used vaccines to control this disease consist of inactivated whole cells (bacterins), whose production cost is high and the efficiency is limited. The objective of this study was to develop and to evaluate in BALB/c mice a recombinant subunit vaccine (rLTBR1) containing the R1 region of P97 adhesin of M. hyopneumoniae (R1) fused to the B subunit of the heat-labile enterotoxin of Escherichia coli (LTB). rLTBR1 formed functional oligomers that presented high affinity to GM1 ganglioside. Mice inoculated with rLTBR1 by intranasal (IN) or intramuscular (IM) route produced high levels of anti-R1 systemic and mucosal antibodies (IgA), which recognized the native P97. On the other hand, mice inoculated with the inactivated whole cell vaccine did not produce anti-R1 antibodies. The administration route influenced the modulation of the immune response by LTB, showing that IM rLTBR1 induced Th2-biased immune responses and IN rLTBR1 induced Th1-biased immune responses. rLTBR1 administrated by IN route also induced IFN-gamma secretion by lymphocytes. rLTBR1 may constitute a new strategy for preventing infection by M. hyopneumoniae and may have potential for developing vaccines against other infectious diseases as well.     

Oral vaccination against mycoplasmal pneumonia of swine using a live Erysipelothrix rhusiopathiae vaccine strain as a vector

Erysipelothrix rhusiopathiae Koganei 65-0.15 strain, the live swine erysipelas vaccine for subcutaneous injection, has been shown to colonize the tonsils of pigs after oral inoculation. We thus evaluated the possible use of the strain as a vector for oral vaccination against mycoplasmal pneumonia of swine. Recombinant E. rhusiopathiae strains expressing the C-terminal domain of the P97 adhesin of Mycoplasma hyopneumoniae were constructed and examined for vaccine efficacy in mice and pigs. Mice subcutaneously inoculated with the recombinant strains were protected from challenge exposure to a virulent E. rhusiopathiae. Administration of milk replacer containing recombinant E. rhusiopathiae expressing the M. hyopneumoniae protein protected pigs from death after exposure to E. rhusiopathiae and significantly reduced the severity of pneumonic lung lesions caused by infection with M. hyopneumoniae.     

Immune responses and protective efficacy of a recombinant swinepox virus expressing HA1 against swine H1N1 influenza virus in mice and pigs

Swine influenza virus (SIV) is not only an important respiratory pathogen in pigs but also a potent threat to human health. Although immunization with recombinant poxviruses expressing protective antigens as vaccines has been widely used for against many infectious diseases, development of recombinant swinepox virus (rSPV) vector for the purpose has been less successful. Here, we report the construction of a recombinant swinepox virus (rSPV-HA1) expressing hemagglutinin (HA1) of H1N1 SIV. Immune responses and protection efficacy of the vaccination vector were evaluated in both the mouse model and the natural host: pig. Prime and boost inoculations of rSPV-HA1 yielded high levels of neutralization antibody against SIV and elicited potent H1N1 SIV-specific IFN-γ response from T-lymphocytes. Complete protection of pigs against H1N1 SIV challenge was observed. No pigs showed evident systemic and local reactions to the vaccine and no SIV shedding was detected from pigs vaccinated with rSPV-HA1 after challenge. Our data demonstrated that the recombinant swinepox virus encoding HA1 of SIV H1N1 may serve as a promising SIV vaccine for protection against SIV infection.     

Enhanced protective efficacy against tuberculosis provided by a recombinant urease deficient BCG expressing heat shock protein 70-major membrane protein-II having PEST sequence

Enhancement of the T cell-stimulating ability of Mycobacterium bovis BCG (BCG) is necessary to develop an effective tuberculosis vaccine. For this purpose, we introduced the PEST-HSP70-major membrane protein-II (MMPII)-PEST fusion gene into ureC-gene depleted recombinant (r) BCG to produce BCG-PEST. The PEST sequence is involved in the proteasomal processing of antigens. BCG-PEST secreted the PEST-HSP70-MMPII-PEST fusion protein and more efficiently activated human monocyte-derived dendritic cells (DCs) in terms of phenotypic changes and cytokine productions than an empty-vector-introduced BCG or HSP70-MMPII gene-introduced ureC gene-depleted BCG (BCG-DHTM). Autologous human naïve CD8⁺ T cells and naïve CD4⁺ T cells were effectively activated by BCG-PEST and produced IFN-γ in an antigen-specific manner through DCs. These T cell activations were closely associated with phagosomal maturation and intraproteasomal protein degradation in antigen-presenting cells. Furthermore, BCG-PEST produced long-lasting memory-type T cells in C57BL/6 mice more efficiently than control rBCGs. Moreover, a single subcutaneous injection of BCG-PEST more effectively reduced the multiplication of subsequent aerosol-challenged Mycobacterium tuberculosis of the standard H37Rv strain and clinically isolated Beijing strain in the lungs than control rBCGs. The vaccination effect of BCG-PEST lasted for at least 6 months. These results indicate that BCG-PEST may be able to efficiently control the spread of tuberculosis in human.     

Evaluation of a recombinant Listeria monocytogenes expressing an HIV protein that protects mice against viral challenge

Vaccine strategies that utilize cell mediated immunity to control infection will be a necessary component of human immunodeficiency virus (HIV) vaccines. In previous studies we have shown that a Listeria monocytogenes recombinant expressing HIV-Gag elicits strong CD8+ and CD4+ T cell responses against HIV Gag in addition to its own secreted proteins. Here, we show that Lm-Gag can protect mice against a viral challenge with a recombinant vaccinia virus expressing Gag, in an antigen specific manner, and that this protection is T cell mediated. These results further support the use of L. monocytogenes as a vaccine approach for HIV through the induction of T cell immunity.     

Preclinical toxicity and efficacy testing of RiVax, a recombinant protein vaccine against ricin

Ricin toxin is a plant-derived ribosome inactivating protein (RIP) of extraordinary toxicity. Vaccination using ricin toxoid or its A chain (RTA) is protective in animals but both vaccines have two potential toxicities, RIP and vascular leak syndrome (VLS). Previously we described three recombinant RTA constructs from which both toxicities were eliminated by site-specific mutations. One mutant, V76M/Y80A, RiVax, has now been further characterized for immunogenicity and toxicity in animals. We have found that RiVax is safe at doses of at least 8 mg in mice, 800-fold higher than the protective dose, and induces neutralizing antibodies in both mice and rabbits.     

Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55 (E2) gene protects pigs against classical swine fever

A recombinant porcine adenovirus (rPAV) with the gp55 (E2) gene from the classical swine fever virus (CSFV) 'Weybridge' strain inserted into the right hand end of the PAV serotype 3 (PAV3) genome was constructed. Expression of gp55 was directed by the major late promoter and tri-partite leader sequences located and cloned from PAV3. No compensatory deletions of PAV DNA sequences were made. Vaccination of outbred pigs with a single dose of the recombinant virus (rPAV-gp55) resulted in complete protection from lethal challenge with CSFV. No adverse clinical signs were observed in vaccinated animals following administration of rPAV-gp55 and following challenge, no clinical signs of CSF were observed prior to, or at, post mortem. The insert made into the rPAV increased the genome length to 106.8% of wild type and therefore exceeded the expected maximum insert size for a stable recombinant by almost 2%. Thus rPAV-gp55 contains the largest stable insertion made into a non-deleted Mastadeno virus recombinant so far reported.     

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.     

Protection of chickens against hepatitis-hydropericardium syndrome and Newcastle disease with a recombinant Newcastle disease virus vaccine expressing the fowl adenovirus serotype 4 fiber-2 protein

Newcastle disease (ND) is one of the most important and devastating avian diseases with considerable threat to the global poultry industry. Hepatitis-hydropericardium syndrome (HHS), caused by virulent fowl adenovirus serotype 4 (FAdV-4), is another highly infectious disease in chickens with severe economic impact. The effective way to combat ND and HHS is by vaccinating the poultry. In the present study, a recombinant NDV LaSota vaccine strain expressing full length fiber-2 gene of FAdV-4 (rLaSota-fiber2) was generated using reverse genetics. The FAdV-4 fiber-2 protein was expressed as a soluble form rather than NDV membrane-anchored form. The rLaSota-fiber2 was genetically stable, and it showed growth patterns in embryonated eggs comparable to that of parental rLaSota virus. Since our unpublished data demonstrated that delivery of live rLaSota-fiber2 in drinking water or ocular delivery of the vaccine didn’t produce protection against hypervirulent FAdV-4 challenge, even though the vaccine provide full protection against NDV challenge, the efficacy of the rLaSota-fiber2 was evaluated by delivering the vaccine intramuscularly in this study. Single-dose intramuscular vaccination of 2-week-old SPF White Leghorn chicks with the live or inactivated rLaSota-fiber2 provided complete protection against virulent NDV challenge. However, single-dose intramuscular vaccination with the live rLaSota-fiber2 vaccine provided better protection against virulent FAdV-4 challenge and significantly reduced faecal viral shedding comparing to the inactivated vaccine. These results indicate that the NDV-vectored FAdV-4 vaccine is a promising bivalent vaccine candidate to control both HHS and ND.     

Co-immunization with interlukin-18 enhances the protective efficacy of liposomes encapsulated recombinant Cu-Zn superoxide dismutase protein against Brucella abortus

Brucellosis is a worldwide zoonotic disease caused by Brucella abortus and a number of closely related species. Brucellosis has severe impact on the health and economic prosperity of the developing countries due to the persistent nature of infection and unavailability of effective control measures. The Cu-Zn superoxide dismuatse (SOD) protein of Brucella have been extensively studied as a major antigen involved in bacterial evading mechanism of host defence. Being a critical pro-inflammatory cytokine interleukin-18 (IL-18) plays key role in induction of immune mediated protection against intracellular pathogens. In the present study, we aimed to investigate the immunogenic potential of fusogenic liposomes (escheriosomes) encapsulated recombinant Cu-Zn SOD (rSOD) protein alone or in combination with recombinant IL-18 (rIL-18). Escheriosomes encapsulated rSOD mediated immune responses were further increased upon co-immunization with rIL-18. Furthermore, immunization with escheriosomes encapsulated rSOD alone or in combination with rIL-18, increased resistance in mice against challenge with B. abortus 544.     

Immunogenicity and protective efficacy of the Mycobacterium avium subsp. paratuberculosis attenuated mutants against challenge in a mouse model

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), results in serious economic losses worldwide especially in cattle, sheep and goats. To control the impact of JD on the animal industry, an effective vaccine with minimal adverse effects is urgently required. In order to develop an effective vaccine, we used allelic exchange to construct three mutant MAP strains, leuD, mpt64 and secA2. The mutants were attenuated in a murine model and induced cytokine responses in J774A.1 cell. The leuD mutant was the most obviously attenuated of the three constructed mutant strains. Our preliminary vaccine trial in mice demonstrated different levels of protection were induced by these mutants based on the acid-fast bacilli burden in livers and spleens at 8 and 12 weeks postchallenge. In addition, vaccination with leuD mutant induced a high level of IFN-γ production and significant protective efficacy in both the reduction of inflammation and clearance of acid-fast bacilli, as compared with the mock vaccinated group.     

Enhanced protection against Rickettsia rickettsii infection in C3H/HeN mice by immunization with a combination of a recombinant adhesin rAdr2 and a protein fragment rOmpB-4 derived from outer membrane protein B

BackgroundTwo surface proteins of Rickettsia rickettsii, outer membrane protein B (OmpB) and adhesion 2 (Adr2), have been recognized as protective antigens. Herein, the immunization with both OmpB and Adr2 was performed in mice so as to explore whether their combination could induce an enhanced immunoprotection against R. rickettsii infection.MethodsC3H/HeN mice were immunized with recombinant protein rAdr2 or/and rOmp-4, a fragment derived from OmpB, and then mice were challenged with R. rickettsii. After which rickettsial loads in mice were measured by quantitative PCR. The specific antibodies in mouse sera were determined by ELISA and antigen-specific cytokines secretion by mouse T cells were analyzed in vitro.ResultsAfter challenge with R. rickettsii, the mice immunized with rAdr2 or/and rOmpB-4 had significant lower rickettsial load in livers, spleens, or lungs compared to PBS mock-immunized mice. Particularly, the load in lungs of mice immunized with both rAdr2 and rOmpB-4 was significantly lower than that with either of them. High levels of specific antibodies were detected in sera from mice immunized with rAdr2 or/and rOmpB-4, but the ratios of specific IgG2a to IgG1 induced by their combination were significantly higher than that by either rAdr2 or rOmpB-4. Following stimulation with rAdr2 or/and rOmpB-4, the INF-γ secreted by CD4⁺ T cells from infected mice was significantly higher than that by cognate cells from uninfected mice. And the TNF-α secreted by CD4⁺ or CD8⁺ T cells from infected mice was markedly greater than that by cognate cells from uninfected mice after stimulation by their combination but not either of them.ConclusionThe combination of rAdr2 and rOmpB-4 conferred an enhanced protection against R. rickettsii infection in mice, which was mainly dependent on a stronger Th1-oriented immunoresponse with greater INF-γ and TNF-α secretion by antigen-specific T cells and specific IgG2a elicited by the combination.     

Improved efficacy of DNA vaccination against prostate carcinoma by boosting with recombinant protein vaccine and by introduction of a novel adjuvant epitope

DNA vaccine represents an attractive approach for cancer treatment by inducing active immune-deprivation of gastrin-releasing peptide (GRP) from tumor cells, the growth of which is dependent on the stimulation of GRP. In this study, we developed a DNA vaccine using a plasmid vector to deliver the immunogen of six copies of the B cell epitope GRP_18–27 (GRP6). In order to increase the potency of this DNA vaccine, multiple strategies have been applied including DNA-prime protein-boost immunization and introduction of a foreign T-helper epitope into DNA vaccine. Mice vaccinated DNA vaccine boosting with HSP65-GRP6 protein induced high titer and relatively high avidity of anti-GRP antibodies as well as inhibition effect on the growth of murine prostate carcinoma, superior to the treatment using DNA alone or BCG priming HSP65-GRP6 protein boosting. Furthermore, the introduction of a novel foreign T-helper epitope into the GRP DNA vaccine showed a markedly stronger humoral immune response against GRP and tumor rejection even than the DNA-prime protein-boost strategy. No further stronger immunogenicity of this foreign T-helper epitope modified DNA vaccine was observed even using the strategy of modified DNA vaccine-priming and HSP65-GRP6 boosting method. The data presented demonstrate that improvement of potency of anti-GRP DNA vaccine with the above two feasible approaches should offer useful methods in the development of new DNA vaccine against growth factors for cancer immunotherapy.     

Alpha interferon is a powerful adjuvant for a recombinant protein vaccine against foot-and-mouth disease virus in swine, and an effective stimulus of in vivo immune response

The adjuvant effect of porcine interferon-alpha (PoIFN-alpha) was examined in swine vaccinated with a recombinant FMD protein vaccine named IgG-FMDV, which contains the swine IgG single heavy chain constant region and an immunogenic peptide of serotype O FMDV. The PoIFN-alpha gene was cloned into pcDNA3 vector and the recombinant plasmid was incorporated into cationic liposomes by a dehydration and rehydration procedure to use as an adjuvant, injected together with low-dose IgG-FMDV. This procedure resulted in strong induction of FMDV-specific neutralizing antibody and significant T-cell-mediated immune responses, whereas only a modest humoral and cellular response was observed with low-dose vaccine alone. As an adjuvant for the protein vaccine, PoIFN-alpha induced strong inflammatory cytokines production in vivo and the results denoted that IFN-adjuvant and our vaccines could drive the immune response toward Th1 type responses. The data of ELISA suggests that the recombinant protein vaccine synergizes with the IFN-adjuvant to produce endogenous IFN in vivo. In response to viral challenge, all control animals developed viremia and lesions, whereas all animals received IFN-adjuvant+IgG-FMDV were protected and nonstructural protein antibody in this group could not be detected by 14 days post-challenge (dpc). Our studies indicate that porcine IFN-alpha is a powerful adjuvant for recombinant FMD protein vaccine and could aid in vaccination against FMDV in swine.     

Protection against murine listeriosis by oral vaccination with recombinant Salmonella expressing protective listerial epitopes within a surface-exposed loop of the TolC-protein

Based on the topology of the outer membrane protein TolC of Escherichia coli, a new plasmid-encoded system was created which allows the expression of antigenic peptides within permissive, surface-exposed domains of TolC. To assess the capacity of this novel antigen display system, a protective CD4 T-cell epitope of the p60 protein of Listeria monocytogenes was inserted within an extracellular loop of the TolC-protein and expressed in surface-exposed form by attenuated Salmonella enteritidis. Mice immunized orally with this recombinant S. enteritidis live vaccine strain were protected against a lethal challenge with wildtype L. monocytogenes.     

Immunogenicity and protective efficacy of a live attenuated vaccine against the 2009 pandemic A H1N1 in mice and ferrets

A novel 2009 influenza A (H1N1) virus was transmitted from humans to humans worldwide. The live attenuated monovalent A H1N1 vaccine (LAMV) for intranasal administration has shown promising immunogenicity and safety in clinical trials and for human use, but the experimental data based on LAMV is incomplete. In this study, using reverse genetic technology, we produced a cold-adapted (ca), live attenuated BJ/AA ca that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2009 pandemic A H1N1 isolate, A/Beijing/501/2009 virus (BJ501), and the remaining six internal gene segments from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). BJ/AA ca exhibited phenotypes of temperature sensitivity (ts), ca, and attenuation (att). The candidate BJ/AA ca was immunogenic in mice and induced strong mucosal secretory IgA (sIgA) in the respiratory tract. Two dosages of intranasal immunization induced robust HI antibodies and offered efficient protection against challenge by the wild-type (wt) 2009 pandemic A H1N1 (A/Beijing/501/2009 or A/California/07/2009) in mice and ferrets. These results support the evaluation of this vaccine made from a wt strain isolated in China for clinical trials.     

Comparison of the protective efficacy of yeast-derived and Escherichia coli-derived recombinant merozoite surface protein 4/5 against lethal challenge by Plasmodium yoelii

The gene encoding the Plasmodium yoelii homologue of P. falciparum merozoite surface proteins 4 (MSP4) and 5 (MSP5) has been expressed in Escherichia coli and Saccharomyces cerevisiae. The protein contains a single epidermal growth factor (EGF)-like domain and is expressed in a form lacking the predicted N-terminal signal and glycosyl phosphatidylinositol (GPI) attachment sequences. The recombinant protein derived from E. coli (EcMSP4/5) was highly effective at protecting mice against lethal challenge with 10⁵ parasites of the P. yoelii YM strain. In contrast, the protective efficacy of yeast-derived MSP4/5 (yMSP4/5) was considerably less. The antibody titres in both groups were significantly different with mice immunised with yeast-derived protein showing significantly lower pre-challenge antibody responses. There was a significant inverse correlation between antibody levels as measured by ELISA and peak parasitaemia. Mice immunised with EcMSP4/5 produced anti-PyMSP4/5 antibodies predominantly of the IgG2a and IgG2b isotypes, whereas, mice immunised with yMSP4/5 mainly produced antibodies of the IgG1 isotype. The differences in antibody titres and subtype distribution may account for the observed differences in protective efficacy of these protein preparations. Levels of protective efficacy of MSP4/5 were compared with that obtained using P. yoelii MSP1 produced in S. cerevisiae. Levels of protection induced by E. coli derived MSP4/5 were superior to those induced by MSP1 which in turn were better than those induced by yeast-derived MSP4/5.     

Immunogenicity and protective efficacy of a recombinant yellow fever vaccine against the murine malarial parasite Plasmodium yoelii

The live-attenuated yellow fever vaccine (YF17D) is one of the safest and most effective vaccines available today. Here, YF17D was genetically altered to express the circumsporozoite protein (CSP) from the murine malarial parasite Plasmodium yoelii. Reconstituted recombinant virus was viable and exhibited robust CSP expression. Immunization of naïve mice resulted in extensive proliferation of adoptively transferred CSP-specific transgenic CD8⁺ T-cells. A single immunization of naïve mice with recombinant YF17D resulted in robust production of IFN-γ by CD8⁺ T-cells and IFN-γ and IL-2 by CD4⁺ T-cells. A prime-boost regimen consisting of recombinant virus followed by a low-dose of irradiated sporozoites conferred protection against challenge with P. yoelii. Taken together, these results show that recombinant YF17D can efficiently express CSP in culture, and prime a protective immune response in vivo.