Alternative Vaccine Candidate Research Articles

Membrane vesicles from Piscirickettsia salmonis induce protective immunity and reduce development of salmonid rickettsial septicemia in an adult zebrafish model

Infections caused by the facultative intracellular bacterial pathogen Piscirickettsia salmonis remains an unsolved problem for the aquaculture as no efficient treatments have been developed. As a result, substantial amounts of antibiotic have been used to limit salmonid rickettsial septicemia (SRS) disease outbreaks. The antibiotic usage has not reduced the occurrence, but lead to an increase in resistant strains, underlining the need for new treatment strategies. P. salmonis produce membrane vesicles (MVs); small spherical structures know to contain a variety of bacterial components, including proteins, lipopolysaccharides (LPS), DNA and RNA. MVs mimics' in many aspects their mother cell, and has been reported as alternative vaccine candidates. Here, MVs from P. salmonis was isolated and evaluated as a vaccine candidate against SRS in an adult zebrafish infection model. When zebrafish was immunized with MVs they were protected from subsequent challenge with a lethal dose of P. salmonis. Histological analysis showed a reduced bacterial load upon challenge in the MV immunized group, and the mRNA expression levels of several immune related genes altered, including mpeg1.1, tnfα, il1b, il10 and il6. The MVs induced the secretion of IgM upon immunization, indicating an immunogenic effect of the vesicles. Taken together, the data demonstrate a vaccine potential of MVs against P. salmonis.

Multi-walled carbon nanotubes functionalized with recombinant Dengue virus 3 envelope proteins induce significant and specific immune responses in mice

BackgroundDengue is the most prevalent arthropod-borne viral disease in the world. In this article we present results on the development, characterization and immunogenic evaluation of an alternative vaccine candidate against Dengue.MethodsThe MWNT-DENV3E nanoconjugate was developed by covalent functionalization of carboxylated multi-walled carbon nanotubes (MWNT) with recombinant dengue envelope (DENV3E) proteins. The recombinant antigens were bound to the MWNT using a diimide-activated amidation process and the immunogen was characterized by TEM, AFM and Raman Spectroscopy. Furthermore, the immunogenicity of this vaccine candidate was evaluated in a murine model.ResultsImmunization with MWNT-DENV3E induced comparable IgG responses in relation to the immunization with non-conjugated proteins; however, the inoculation of the nanoconjugate into mice generated higher titers of neutralizing antibodies. Cell-mediated responses were also evaluated, and higher dengue-specific splenocyte proliferation was observed in cell cultures derived from mice immunized with MWNT-DENV3E when compared to animals immunized with the non-conjugated DENV3E.ConclusionsDespite the recent licensure of the CYD-TDV dengue vaccine in some countries, results from the vaccine’s phase III trial have cast doubts about its overall efficacy and global applicability. While questions about the effectiveness of the CYD-TDV vaccine still lingers, it is wise to keep at hand an array of vaccine candidates, including alternative non-classical approaches like the one presented here.

Protection Elicited by Nasal Immunization with Recombinant Pneumococcal Surface Protein A (rPspA) Adjuvanted with Whole-Cell Pertussis Vaccine (wP) against Co-Colonization of Mice with Streptococcus pneumoniae.

A promising alternative vaccine candidate to reduce the burden of pneumococcal diseases is the protein antigen PspA (Pneumococcal surface protein A). Since concomitant colonization with two or more pneumococcal strains is very common in children, we aimed to determine if immunization with PspA would be able to control co-colonization. We evaluated nasal immunization with recombinant PspA (rPspA) in a model of co-colonization with two strains expressing different PspAs. Mice were immunized intranasally with rPspAs from clades 1 to 4 (rPspA1, rPspA2, rPspA3 or rPspA4) using whole-cell pertussis vaccine (wP) as adjuvant. Mice were then challenged with a mixture of two serotype 6B isolates St491/00 (PspA1) and St472/96 (PspA4). Immunization with rPspA1+wP and rPspA4+wP reduced colonization with both strains and the mixture of rPspA1+rPspA4+wP induced greater reduction than a single antigen. Immunization rPspA1+rPspA4+wP also reduced colonization when challenge experiments were performed with a mixture of isolates of serotypes 6B (PspA3) and 23F (PspA2). Furthermore, none of the tested formulations led to a pronounced increase in colonization of one isolate over the other, showing that the vaccine strategy would not favor replacement. Interestingly, the adjuvant wP by itself already led to some reduction in pneumococcal colonization, indicating the induction of non-specific immune responses. Anti-rPspA IgG was observed in serum, nasal wash (NW) and bronchoalveolar lavage fluid (BALF) samples, whereas animals inoculated with formulations containing the adjuvant wP (with or without rPspA) showed higher levels of IL-6 and KC in NW and increase in tissue macrophages, B cells and CD4+T cells in BALF.

Structural and Immunological Characterization of Recombinant 6-Cysteine Domains of the Plasmodium falciparum Sexual Stage Protein Pfs230

Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel® appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine. Pfs230, a 230-kDa sexual stage protein expressed in gametocytes is an alternative vaccine candidate. A unique 6-cysteine-rich domain structure within Pfs230 have thwarted its recombinant expression and characterization for clinical evaluation for nearly a quarter of a century. Here, we report on the identification, biochemical, biophysical, and immunological characterization of recombinant Pfs230 domains. Rabbit antibodies generated against recombinant Pfs230 domains blocked mosquito transmission of a laboratory strain and two field isolates using an ex vivo assay. A planned clinical trial of the Pfs230 vaccine is a significant step toward the potential development of a transmission blocking vaccine to eliminate malaria.

Meningococcal vaccine antigen diversity in global databases.

The lack of an anti-capsular vaccine against serogroup B meningococcal disease has necessitated the exploration of alternative vaccine candidates, mostly proteins exhibiting varying degrees of antigenic variation. Analysis of variants of antigen-encoding genes is facilitated by publicly accessible online sequence repositories, such as the Neisseria PubMLST database and the associated Meningitis Research Foundation Meningococcus Genome Library (MRF-MGL). We investigated six proposed meningococcal vaccine formulations by deducing the prevalence of their components in the isolates represented in these repositories. Despite high diversity, a limited number of antigenic variants of each of the vaccine antigens were prevalent, with strong associations of particular variant combinations with given serogroups and genotypes. In the MRF-MGL and globally, the highest levels of identical sequences were observed with multicomponent/multivariant vaccines. Our analyses further demonstrated that certain combinations of antigen variants were prevalent over periods of decades in widely differing locations, indicating that vaccine formulations containing a judicious choice of antigen variants have potential for long-term protection across geographic regions. The data further indicated that formulations with multiple variants would be especially relevant at times of low disease incidence, as relative diversity was higher. Continued surveillance is required to monitor the changing prevalence of these vaccine antigens.

Rationale and prospects for novel pneumococcal vaccines

Streptococcus pneumoniae remains one of the most frequent bacterial causes of morbidity and mortality worldwide. National immunization programs implementing pneumococcal polysaccharide conjugate vaccines (PCVs) have successfully reduced rates of vaccine-type invasive disease and colonization both via direct effects in immunized children and, in some settings, indirect effects in unimmunized individuals. Limitations of the current PCV approach include the emergence of non-vaccine serotypes contributing to carriage and invasive disease in high-PCV coverage settings and the high cost of goods of PCVs which limits their accessibility in developing countries where the burden of disease remains highest. Furthermore, the distribution of serotypes causing disease varies geographically and includes more serotypes than are currently covered in a single PCV formulation. Researchers have long been exploring the potential of genetically conserved non-capsular pneumococcal antigens as vaccine candidates that might overcome such limitations. To better evaluate the rationale of such approaches, an understanding of the mechanisms of immunity to the various phases of pneumococcal infection is of paramount importance. Herein we will review the evolving understanding of both vaccine-induced and naturally acquired immunity to pneumococcal colonization and infection and discuss how this informs current approaches using serotype-independent pneumococcal vaccine candidates. We will then review the alternative vaccine candidates that have been or are currently under evaluation in clinical trials.

Immunogenicity of porcine P[6], P[7]-specific △VP8* rotavirus subunit vaccines with a tetanus toxoid universal T cell epitope

Currently, commercial porcine rotavirus vaccines remain varied limitations. The objective of this study is to develop an alternative porcine rotavirus subunit vaccine candidate by parenteral administration, which enables to elicit robust immune responses against most prevalence porcine rotavirus strains. The bacterially-expressed porcine rotavirus P[6]- or P[7]-specific truncated VP8* (aa 64–223) recombinant protein with or without a universal tetanus toxoid CD4+ T cell epitope P2 was generated. All the recombinant subunit proteins △VP8*s or P2-△VP8*s were of high solubility and high yields. The immunogenicity of each purified △VP8* and P2-△VP8* was evaluated in mice (10μg/dose) or guinea pigs (20μg/dose) immunized IM with 600μg aluminum hydroxide three times at 2-week interval. The introduction of P2T cell epitope to P[7]-△VP8* elicited significantly higher IgG titer in mice than its absence. Comparatively, P2 epitope slightly enhanced the immunogenicity of P[6]-△VP8*. P2-P[7]△VP8* elicited high titer of neutralizing antibody against heterotypic P[7]-specific rotaviruses with varied G type combination. Our data indicated that two subunit vaccines could be plausible bivalent rotavirus vaccine candidate to provide antigenic coverage of porcine rotavirus strains of global or regional importance.

Chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in Africa.

Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented. Thus, it is now possible to generate tRV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain.

Detoxified pneumolysin derivative plym2 directly protects against pneumococcal infection via induction of inflammatory cytokines

Streptococcus pneumoniae is a major cause of infectious disease and complications worldwide, such as pneumonia, otitis media, bacteremia and meningitis. New generation protein-based pneumococcal vaccines are recognized as alternative vaccine candidates. Pneumolysin (Ply) is a cholesterol-dependent cytolysin produced by all clinical isolates of S. pneumoniae. Our research group previously developed a highly detoxified Ply mutant designated Plym2 by replacement of two animo acids (C428G and W433F). Exhibiting undetectable levels of cytotoxicity, Plym2 could still elicit high titer neutralizing antibodies against the native toxin. However, evaluation of the active immunoprotective effects of Plym2 by subcutaneous immunization and lethal challenge with S. pneumoniae in mice did not yield favorable results. In the present work, we confirmed the previous observations by using passive immunization and systemic challenge. Results of the passive immunization were consistent with those of active immunization. Further experiments were conducted to explain the inability of high titer neutralizing antibodies against Ply to protect mice from S. pneumoniae challenge. Pneumococcal Ply is known to be the major factor responsible for the induction of inflammation that benefits the host. Proinflammatory cytokines facilitate the clearance of invaders by the recruitment and activation of leukocytes at the early infection stage. We demonstrated that Plym2 could induce proinflammatory cytokines similarly to wild-type Ply. A systemic infection model was used to clarify that Plym2 lacking cytolytic activity could protect mice from intraperitoneal challenge directly, while antibodies to the mutant had no effect. Therefore, the protective function of Plym2 may be due to its induction of proinflammatory cytokines. When used in the systemic infection model, Plym2 antibodies may block the induction of proinflammatory cytokines by Ply. These findings demonstrate that a Ply-based vaccine would not be an effective primary vaccine component, but it may be beneficial as an adjuvant to stimulate cytokine production.

Possibilities and limitations in veterinary vaccine development using the example of classical swine fever

The use of vaccines is still one of the most effective tools to control infectious diseases. Up to now, conventional vaccines are employed in the majority of cases. Drawbacks of these established vaccines include the lack of differentiability of infected from vaccinated animals (DIVA or marker strategy), limitations in the efficacy spectrum, and constraints and restrictions in production. For this reason, new vaccines, which do not show these disadvantages, are under development, especially for notifiable diseases such as classical swine fever (CSF). In principle, the following modern vaccine types can be differentiated: recombinant attenuated vaccines, recombinant inactivated vaccines or subunit vaccines, vector vaccines, and DNA/ RNA vaccines. During the last years, especially attenuated deletion vaccines or chimeric constructs have shown potential. Under field conditions, all marker vaccines have to be accompanied by a potent test system. Particularly this point often shows weaknesses. Alternative vaccine candidates are so far only prototypes and licensing is only a medium term possibility. Moreover, most of these vaccines are genetically engineered and can be problematic in terms of licensing and the public's acceptance. In conclusion, conventional vaccines still present the standard, especially in terms of efficacy. Yet, only vaccines with DIVA properties are feasible for the control of CSF. Thus, development and assessment of alternative vaccines is of paramount importance. The present overview summarizes concepts and vaccine types using the example of classical swine fever. It also recapitulates their advantages and disadvantages as well as their limitations.

Identification of Potential Universal Vaccine Candidates against Group A Streptococcus by Using High Throughput in Silico and Proteomics Approach

Streptococcus pyogenes or group A Streptococcus (GAS) causes ~700 million human infections each year, resulting in over 500,000 deaths. The development of a commercial GAS vaccine is hampered due to high strain and serotype diversity in different geographical regions, and the generation of cross-reactive antibodies that may induce autoimmune disease. There is an urgent need to search for alternative vaccine candidates. High throughput multigenome data mining coupled with proteomics seems to be a promising approach to identify the universal vaccine candidates. In the present study, in silico analysis led to prediction of 147 proteins as universal vaccine candidates. Distribution pattern of these predicted candidates was explored in nonsequenced Indian GAS strains (n = 20) by using DNA array hybridization validating in silico analysis. High throughput analyses of surface proteins using 1D-SDS-PAGE coupled with ESI-LC-MS/MS was applied on highly (M49) and less (M1) invasive GAS strains of Indian origin. Comparative proteomics analysis revealed that highly invasive GAS M49 had metabolically more active membrane associated protein machinery than less invasive M1. Further, by overlapping proteomics data with in silico predicted vaccine candidate genes, 52 proteins were identified as probable universal vaccine candidates, which were expressed in these GAS serotypes. These proteins can further be investigated as universal vaccine candidates against GAS. Moreover, this robust approach may serve as a model that can be applied to identify the universal vaccine candidates in case of other pathogenic bacteria with high strain and genetic diversity.

Variation and molecular evolution of HmbR, the Neisseria meningitidis haemoglobin receptor

Meningococcal disease caused by serogroup B Neisseria meningitidis remains an important health problem in many parts of the world, and there are currently no comprehensive vaccines. Poor immunogenicity, combined with immunological identity to human sialic acids, have hindered the development of a serogroup B conjugate vaccine, resulting in the development of alternative vaccine candidates, including many outer-membrane protein (OMP)-based formulations. However, the design of protein-based meningococcal vaccines is complicated by the high level of genetic and antigenic diversity of the meningococcus. Knowledge of the extent and structuring of this diversity can have implications for the use of particular proteins as potential vaccine candidates. With this in mind, the diversity of the meningococcal OMP HmbR was investigated among N. meningitidis isolates representative of major hyper-invasive lineages. In common with other meningococcal antigens, the genetic diversity of hmbR resulted from a combination of intraspecies horizontal genetic exchange and de novo mutation. Furthermore, genealogical analysis showed an association of hmbR genes with clonal complexes and the occurrence of two hmbR families, A and B. Three variable regions (VR1–VR3), located in loops 2, 3 and 4, were observed with clonal complex structuring of VR types. A minority of codons (3.9 %), located within putative surface-exposed loop regions of a 2D model, were under diversifying selection, indicating regions of the protein likely to be subject to immune attack.

Vaccine development through terminal deletions of an infectious bursal disease virus protein 2 precursor variant

VP2 is the primary host-protective immunogen of infectious bursal disease virus (IBDV), the agent that causes the highly contiguous infectious bursal disease (IBD). Previous studies have shown that a C-terminal his-tagged 452 amino acid residue VP2 precursor variant (VP2-452H) can form an immunogenic subviral particle (SVP). A set of his-tagged N- and C-terminal VP2-452 deleting mutants (designated as N5-452H, N10-452H, N20-452H, N40-452H, VP2-441H, VP2-437H, VP2-411H and VP2-399H) was expressed in insect cells to discover the role of both N- and C-termini on the assembly of SVP and to develop an efficient SVP-based vaccine. Among these mutants, the expression level of N5-452H was the highest. Results of ultracentrifugation and electron microscopy also indicated that mutants of N-terminal deletion N10-452H, N20-452H and N40-452H or C-terminal deletion VP2-411H and VP2-399H lost the capability to self-assemble SVP. The other mutants, N5-452H, VP2-441H and VP2-437H, formed SVP. Additionally, SVP formed by N5-452H could not only be single-step purified by immobilized metal-ion affinity chromatography (IMAC), but it could also induce a high titer of neutralizing activity to protect chicks from the infection of IBDV at a low dosage (0.2 μg), suggesting that SVP formed by N5-452H can be an alternative vaccine candidate for the prevention of IBD.

Cell‐Mediated Immune Responses and Protective Efficacy against Infection with Mycobacterium tuberculosis Induced by Hsp65 and hIL‐2 Fusion Protein in Mice

Heat shock protein 65 (Hsp65) is an important immunodominant antigen against tuberculosis (TB), and interleukin-2 (IL-2) plays an important role in the regulation of antimycobacteria immune responses. In order to further increase the immunogenicity of Hsp65 against infection caused by Mycobacterium tuberculosis (MTB), we expressed MTB Hsp65 and human IL-2 fusion protein, Hsp65-hIL-2, in Escherichia coli. The expression of Hsp65-hIL-2 was confirmed by Western blotting using anti-Hsp65 MoAb and anti-hIL-2 MoAb, respectively. Hsp65-IL-2 and Hsp65 were then purified by Ni-NTA affinity chromatography. Mice were immunized with purified Hsp65-hIL-2 or Hsp65 emulsified in the adjuvant combination dimethyl dioctadecylammonium bromide and monophosphoryl lipid A. Eight weeks after immunization, there was significant proliferation of spleen lymphocytes in response to both Hsp65 and Hsp65-hIL-2 proteins. Interestingly, Hsp65-hIL-2 fusion protein elicited significantly higher levels of IFN-gamma and IL-2 in the lymphocytes culture supernatant than that of the BCG (Denmark strain) immunized group and Hsp65 group (P < 0.05). After challenging the immunized mice with MTB, the bacteria loads in the spleens and lungs of mice immunized with the fusion protein were significantly lower than Hsp65 alone group, reaching an equivalent level as BCG immunization group. Our results suggest that the Hsp65 and hIL-2 fusion protein may serve as an alternative vaccine candidate against MTB infection.

Enhanced and Enduring Protection against Tuberculosis by Recombinant BCG-Ag85C and Its Association with Modulation of Cytokine Profile in Lung

BackgroundThe variable efficacy (0–80%) of Mycobacterium bovis Bacille Calmette Guréin (BCG) vaccine against adult tuberculosis (TB) necessitates development of alternative vaccine candidates. Development of recombinant BCG (rBCG) over-expressing promising immunodominant antigens of M. tuberculosis represents one of the potential approaches for the development of vaccines against TB.Methods/Principal FindingsA recombinant strain of BCG - rBCG85C, over expressing the antigen 85C, a secretory immuno-dominant protein of M. tuberculosis, was evaluated for its protective efficacy in guinea pigs against M. tuberculosis challenge by aerosol route. Immunization with rBCG85C resulted in a substantial reduction in the lung (1.87 log10, p<0.01) and spleen (2.36 log10, p<0.001) bacillary load with a commensurate reduction in pathological damage, when compared to the animals immunized with the parent BCG strain at 10 weeks post-infection. rBCG85C continued to provide superior protection over BCG even when post-challenge period was prolonged to 16 weeks. The cytokine profile of pulmonary granulomas revealed that the superior protection imparted by rBCG85C was associated with the reduced levels of pro-inflammatory cytokines - interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, moderate levels of anti-inflammatory cytokine - transforming growth factor (TGF)-β along with up-regulation of inducible nitric oxide synthase (iNOS). In addition, the rBCG85C vaccine induced modulation of the cytokine levels was found to be associated with reduced fibrosis and antigen load accompanied by the restoration of normal lung architecture.Conclusions/SignificanceThese results clearly indicate the superiority of rBCG85C over BCG as a promising prophylactic vaccine against TB. The enduring protection observed in this study gives enough reason to postulate that if an open-ended study is carried out with low dose of infection, rBCG85C vaccine in all likelihood would show enhanced survival of guinea pigs.

Current use and development of vaccines for Japanese encephalitis

Background: Japanese encephalitis (JE) is a significant cause of human morbidity and mortality throughout Asia. Vaccines for JE have been available for many years and their use has been effective in reducing the incidence of JE disease in several countries but, as disease incidence has decreased, concerns regarding adverse events following immunisation have increased. Objective: To review existing JE vaccines and new candidates in advanced preclinical or clinical evaluation. Methods: The review primarily covers published and some unpublished literature from the past decade describing current use of approved JE vaccines in various parts of the world, and advanced development and clinical testing of alternative vaccine candidates. Results/conclusion: There is a clear need for additional licensing of existing or new JE vaccines. Several promising candidates are currently in use or completing clinical trials.

Recombinant Modified Vaccinia Virus Ankara–Based Vaccine Induces Protective Immunity in Mice against Infection with Influenza Virus H5N1

Since 2003, the number of human cases of infections with highly pathogenic avian influenza viruses of the H5N1 subtype is still increasing, and, therefore, the development of safe and effective vaccines is considered a priority. However, the global production capacity of conventional vaccines is limited and insufficient for a worldwide vaccination campaign. In the present study, an alternative H5N1 vaccine candidate based on the replication-deficient modified vaccinia virus Ankara (MVA) was evaluated. C57BL/6J mice were immunized twice with MVA expressing the hemagglutinin (HA) gene from influenza virus A/Hongkong/156/97 (MVA-HA-HK/97) or A/Vietnam/1194/04 (MVA-HA-VN/04). Subsequently, recombinant MVA-induced protective immunity was assessed after challenge infection with 3 antigenically distinct strains of H5N1 influenza viruses: A/Hongkong/156/97, A/Vietnam/1194/04, and A/Indonesia/5/05. Our data suggest that recombinant MVA expressing the HA of influenza virus A/Vietnam/1194/04 is a promising alternative vaccine candidate that could be used for the induction of protective immunity against various H5N1 influenza strains.

The Immunogenicity of a Conformationally Restricted Peptide Mimetic of Meningococcal Lipooligosaccharide

Life-threatening meningitis and septicaemia caused by Neisseria meningitidis are a public health priority, and their prevention by vaccination is a major objective. Meningococcal capsular polysaccharide-based vaccines are effective against the major invasive serogroups, except for serogroup B, the capsule of which mimics human polysaccharides and is poorly immunogenic. An alternative vaccine candidate that has the potential to offer cross-protection against antigenically diverse meningococci is the lipooligosaccharide (LOS). The structurally constrained peptide mimetic, C22, of a bactericidal antibody epitope within LOS was previously shown to elicit cross-reactive antibodies to meningococcal LOS when complexed to NeutrAvidintrade mark as a carrier protein. The immunogenicity of this antigen in H-2(d) (BALB/c) and H-2(k) (C3H/HeN) haplotype mice was further investigated. Anti-LOS immunoglobulin G (IgG) antibody titres increased with the vaccine dose and correlated with the anti-C22 peptide antibody titres in both haplotypes. Antigen-stimulated Th1/Th2 cytokine secretion by splenocytes and antibody isotypes indicated a Th2-type immune response with IgG1 antibodies and a low titre of IgG2b. There was no serum bactericidal activity observed against the meningococcus.

Characterization of particles formed by the precursor protein VPX of infectious bursal disease virus in insect Hi-5 cells: implication on its proteolytic processing

The precursor (VPX) of host immunogen VP2 protein for infectious bursal disease virus (IBDV) was expressed in insect Sf9 and Hi-5 cells, and the types of particles generated as well as the immunogenicity induced by these particles were examined. Recombinant VPXH (rVPXH) protein, expressed in Hi-5 cells at an expression level 4× higher than in Sf9 cells, was efficiently processed by proteases to yield VP2-like proteins with corresponding molecular weight, a phenomenon not observed previously. At least three structures of particles were observed for VPXH and VP2-like proteins purified by immobilized metal–ion affinity chromatography (MAC). In addition to the two previously identified twisted tubular and isometric particle structures, there was a new one: icosahedral particles of approximately 25 nm in diameter. The purified particles were further separated by gel-filtration chromatography (GFC) linking with HPLC, which was able to resolve the isometric from icosahedral particles better than ultracentrifugation. Chromatographic results indicate that rVPXH protein mainly involved in the formation of the isometric particle structure and occasionally twisted tubular structure, and the icosahedral particles were formed by the degraded products of rVPXH (VP2-like proteins). Thus, by combining IMAC and GFC, it was shown that VPX was processed efficiently to yield VP2-like protein that could form small virus-like particles in Hi-5 cells. Finally, we demonstrated that virus-neutralizing antibodies were induced when susceptible chickens were vaccinated with the IMAC-purified rVPXH protein (40 μg per bird). This indicates that these particles are highly immunogenic and might serve as an alternative vaccine candidate for the development of IBDV subunit vaccine.

Pneumococcal vaccines: an update on current strategies

Streptococcus pneumoniae is a major cause of morbidity and mortality in infants, children and the elderly. Despite the availability of excellent antimicrobial therapy and adequate health care systems, respiratory diseases and invasive infections caused by pneumococci still comprise a major health problem. The emerging resistance to penicillin and other commonly used antibiotics underscores the importance of the development of novel vaccine strategies to combat pneumococcal disease. Although the 23-valent polysaccharide (PS) vaccine is immunogenic and protective in most adults and children over 5 years of age, they fail to protect children under 2 years of age. Fortunately, the recent conjugate vaccines have shown to be highly efficacious in preventing invasive diseases in this risk group. Moreover, promising results regarding prevention of pneumonia and acute otitis media have been published. Unfortunately, protection is raised against a limited number of pneumococcal serotypes, and serotype replacement and subsequent vaccine failure have become a serious concern. Currently, several pneumococcal surface proteins are considered as alternative vaccine candidates because of their serotype-independence. Thus far, pneumococcal surface adhesin A (PsaA) has proven to be highly protective against colonization in animal models. Moreover, pneumococcal surface protein A (PspA) and pneumolysin have shown to elicit protection against invasive diseases. Future research will elucidate their true potential in protecting humans. In this paper we discuss the present knowledge on pneumococcal vaccines and the current status of novel vaccine strategies.