Development of recombinant vaccines against bluetongue

Bluetongue virus is the aetiological agent of bluetongue, a disease of domestic and wild ruminants. Twenty-four serotypes are recognized. Novel subunit vaccines, that complement existing modified live polyvalent vaccines, are being developed. Serotype-specific viral neutralizing antibodies that are able to protect sheep against virulent homologous virus challenge can be induced by immunizing with the BTV outer capsid protein VP2 purified from virions or with VP2 expressed by baculovirus recombinants. Presentation of VP2 on virus-like particles, which assemble upon co-expression of the four major structural viral proteins (VP2, VP5, VP3 and VP7), improves the protective effect of VP2. Sheep immunized with core-like particles, comprised of VP3 and VP7, developed only limited clinical signs after virulent virus challenge, demonstrating that not only the outer capsid proteins, but also the core proteins are involved in protection against bluetongue.     

Application of recombinant hemagglutinin proteins as alternative antigen standards for pandemic influenza vaccines

Objectives

The single radial immunodiffusion (SRID) assay, used to quantify hemagglutinin (HA) in influenza vaccines, requires reference reagents; however, because centralized production of reference reagents may slow the emergency deployment of vaccines, alternatives are needed.

Results

We investigated the production of HA proteins using recombinant DNA technology, rather than a traditional egg-based production process. The HA proteins were then used in an SRID assay as a reference antigen. We found that HA can be quantified in both egg-based and cell-based influenza vaccines when recombinant HAs (rHAs) are used as the reference antigen. Furthermore, we confirmed that rHAs obtained from strains with pandemic potential, such as H5N1, H7N3, H7N9, and H9N2 strains, can be utilized in the SRID assay. The rHA production process takes just one month, in contrast to the traditional process that takes three to four months.

Conclusions

The use of rHAs may reduce the time required to produce reference reagents and facilitate timely introduction of vaccines during emergencies.

     

Attenuated vaccines for tropical theileriosis, babesiosis and heartwater: the continuing necessity

Overwhelming evidence has accumulated of the effectiveness of immunization with live attenuated vaccines to control tick-borne diseases of livestock. Despite several disadvantages, vaccination with live attenuated organisms against tropical theileriosis, babesiosis and possibly heartwater constitutes one of the most cost-effective intervention strategies. Although great advances have been made through genomics and proteomics research, this has not yet translated into effective non-living vaccines. As a result, there is a continuing necessity to use available live vaccines in tick and tick-borne disease-control strategies adapted to conditions prevailing in many parts of the world.     

In vitro antigen ELISA for quality control of tetanus vaccines

Consistency of production is recognised as an important aspect of vaccine manufacture and suitably validated in vitro assays are required for quality control testing of these products. For the manufacture and batch release of tetanus vaccines, antigen content and integrity, and degree of adsorption of antigen to the adjuvant are critical parameters that should be monitored for consistency. Here we describe the development and use of an Enzyme Linked Immunosorbent Assay (ELISA) to quantify tetanus antigen in combined vaccine products and to measure the degree of adsorption of antigen to adjuvant. Whilst the antigen assay cannot be assumed to predict potency for different products, it can be used as part of a panel of in vitro methods to provide a more informative product profile and to monitor trends in production. The antigen assay is particularly valuable for providing quantitative information on every final lot when modifications of in vivo potency tests, such as single dilution assays, are used.     

Live recombinant vaccines for humans

Vaccines are clearly the most effective means of preventing infectious diseases and have been particularly successful in controlling viral infection. For example, global small-pox eradication has been the greatest achievement in this regard. However, many existing vaccines are not efficient and there are many diseases against which vaccines are not available at all.     

Tropical theileriosis in Tunisia: epidemiology and control

In Tunisia, tropical theileriosis (Theileria annulata infection of cattle) is one of the major diseases affecting cattle in the summer. Each year about 2,500 clinical cases are recorded in the country, mainly in pure-bred animals. An attempt was made in the North of Tunisia to estimate the direct cost related to clinical cases of TT. In the endemic regions, three states of endemicity were described according to cattle age categories at highest disease risk: (i) endemic stability, (ii) low endemic instability, (iii) high endemic instability. The characterisation of these endemic situations, which are conditioned by the levels of the vector tick population and the quantitative aspects of the infection in ticks, are highly relevant for the development of a live attenuated cell line vaccine against TT targeting the cattle population at disease risk in Tunisia. A research programme was set up to develop the attenuation of four local parasite stocks. Two cell lines infected with two distinct T. annulata stocks were tested in the field on pure-bred animals of different age groups from regions with high disease incidence. The vaccination with each of the two cell lines showed a high efficacy. However significant differences in vaccine reactions rates were observed between the two attenuated cell lines emphasising the importance of achieving an optimal balance between protection and vaccine tolerance particularly in pure-bred lactating cows.     

Differing T-cell requirements for recombinant retrovirus vaccines.

Friend murine leukemia virus is a retrovirus complex that induces rapid erythroleukemia and immunosuppression in susceptible strains of adult mice. Using this model, we directly examined the T-cell subsets required for a protective retrovirus vaccine. Paradoxically, recovery in mice immunized with a chimeric envelope containing only T-helper (TH) and B-cell epitopes was dependent on CD8+ T cells as well as CD4+ T cells despite the fact that the vaccine contained no CD8+ cytolytic T-lymphocyte (CTL) epitopes. However, the requirement for CD8+ T cells was overcome by inclusion of additional TH and B-cell epitopes in the immunizing protein. These additional epitopes primed for more rapid production of virus-neutralizing antibody which appeared to limit virus spread sufficiently to protect even in the absence of CD8+ T cells. Inclusion of an immunodominant CTL epitope in the vaccine was not sufficient to overcome dependence on CD4+ T cells. These data suggest that TH priming is more critical for retrovirus immunity than CTL priming.     

The use of vaccinia virus for the construction of recombinant vaccines

Recombinant DNA technology has been used to engineer vaccinia virus genetically into a eukaryotic expression vector. An exciting outcome of these gene-splicing techniques is that after the insertion of one or more genes which encode the information for antigens responsible for conferring immunity toward an infectious disease, vaccinia can be adapted for the development of live recombinant vaccines. This review discusses recombinant vaccinia design and the feasibility of using these vaccines for disease protection.     

Immune responses dependent on antigen location in recombinant attenuated Salmonella typhimurium vaccines following oral immunization

The subcellular location of a recombinant antigen in recombinant attenuated Salmonella vaccines may influence immunogenicity dependent on exposure of the recombinant antigen to cells involved in systemic immune responses. It has been shown that a recombinant attenuated Salmonella vaccine secreting the recombinant Streptococcus pneumoniae PspA (rPspA) antigen specified by pYA3494 induced protective anti-rPspA-specific immune responses (Kang et al. (2002) Infect. Immun. 70, 1739-1749). A recombinant plasmid pYA3496 specifying a His(6)-tagged rPspA (His(6)-rPspA) protein (no apparent signal sequence) caused the rPspA antigen to localize to the cytoplasm of Salmonella. Salmonella vaccines carrying pYA3494 or pYA3496 expressed similar amounts of rPspA. After a single oral immunization in BALB/c mice with 10(9) colony-forming units (CFU) of the recombinant Salmonella vaccines carrying pYA3494 or pYA3496, IgG antibody responses were stimulated to both rPspA and Salmonella lipopolysaccharide (LPS) antigens. The anti-rPspA IgG titer induced by Salmonella carrying pYA3494 (1.9 x 10(7)) was 10(4) times higher than induced by Salmonella carrying pYA3496 (<2.4 x 10(3)).     

Using recombinant DNA technology for the development of live-attenuated dengue vaccines

Dramatic increases in dengue (DEN) incidence and disease severity have been reported, in great part due to the geographic expansion of Aedes aegypti and Aedes albopictus mosquitoes. One result is the expanded co-circulation of all dengue 1-4 serotype viruses (DENV) in urban areas worldwide, especially in South and South-East Asia, and South America. DEN disease severity ranges from asymptomatic infections to febrile dengue fevers (DF) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). There is an urgent need for a safe and effective tetravalent DEN vaccine. Several live attenuated, tetravalent DEN vaccine candidates have been generated by recombinant DNA technology; these candidates are capable of providing immunity to all four DENV serotypes. In this paper we review (a) recombinant live-attenuated DEN vaccine candidates in terms of deletion, antigen chimerization, and the introduction of adaptive mutations; (b) strategies for improving tetravalent vaccine attenuation; and (c) live-attenuated DENV vaccine development.     

Soluble recombinant influenza vaccines.

Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the neuraminidase gene to a tetramerizing leucine zipper sequence; the resulting product was enzymatically active, tetrameric neuraminidase. The protective immunity induced by this engineered neuraminidase, however, remained fairly strain-specific. A third influenza A virus protein, the M2 protein, has only 23 amino acids exposed on the outer membrane surface. This extracellular part, M2e, has been remarkably conserved in all human influenza A strains since 1933. By fusing the M2e sequence to hepatitis B virus core protein, we could obtain highly immunogenic particles that induced complete, strain-independent, long-lasting protection in mice against a lethal viral challenge. Native M2 is a tetrameric protein and this conformation of the M2e part can also be mimicked by fusing this sequence to a tetramerizing leucine zipper. The potential of the resulting protein as a vaccine candidate remains to be evaluated.     

Strategies for cancer therapy using carcinoembryonic antigen vaccines

Advances in molecular biology and immunology have renewed interest in the development of vaccines for the treatment or prevention of cancer. Research over the past 10 years has focused on the identification of suitable tumour antigens to use as targets for a variety of vaccine strategies. Carcinoembryonic antigen (CEA) was one of the first tumour antigens described, and is commonly expressed by a wide range of adenocarcinomas. Recent studies have identified several human-leukocyte-antigen-restricted epitopes (short peptides) within the CEA protein that can be recognised by human T lymphocytes (T cells). Although CEA-expressing tumour cells are generally weakly recognised by the immune system, several new strategies have been used to enhance immune responses against CEA. This includes using antibodies directed against CEA; inserting the CEA gene into recombinant viruses and bacteria as viral and bacterial vaccines; pulsing the CEA protein, peptides, DNA or RNA onto dendritic cells (specialised antigen-presenting cells); and combining CEA vaccines with cytokines or co-stimulatory molecules to increase vaccine effectiveness. Other factors that might be important in establishing systemic immunity against CEA are the dose, route, timing, and choice of vector and adjuvants for vaccine administration. Further research in understanding the fundamental processes involved in tumour-cell recognition by the immune system, better animal models, and improved clinical trial designs will help to define the full potential of CEA as a target for cancer vaccine development.     

Perspectives for subunit vaccines for the control of ticks

For the first time, successful vaccination against a tick has been carried out using a single defined antigen. Further, it has been shown that it is feasible to produce active antigenic material by recombinant DNA technology. This represents a significant advance towards the development of an alternative means of tick control. Nevertheless, as with any new product and new technology, much developmental work still has to be done before one can be confident that a practical means of tick control will result. From the published information, it does not seem that research on vaccines against other tick species is as advanced as that on Boophilus microplus. The work on B. microplus may, however, provide a short cut to the development of further tick vaccines.     

Experimental molecular design of recombinant vaccines

A method was elaborated to construct combined artificial immunogens mimicking virus particles. The gist was exposing protein antigenic determinants of one virus on the particle surface and delivering plasmids with genes for antigenic proteins of another virus to specialized immune cells. Such immunogens were constructed and shown to induce biosynthesis of specific antibodies against HIV-1 and the tick-borne encephalitis virus. The level and duration of the humoral and cell responses were assayed.     

Development of cancer vaccines using autologous and recombinant high molecular weight stress proteins

High molecular weight heat shock proteins (HSPs), hsp110 and grp170, derived from cancer cells have been previously shown to elicit tumor-specific immunity. This phenomenon is attributed to the antigenic peptides associated with the HSPs. Based on the unique chaperoning properties of these HSPs, a new vaccination strategy has been recently developed to elicit antigen-specific antitumor immunity. This approach utilizes tumor-associated antigens naturally complexed to these highly efficient molecular chaperones under heat shock conditions. This chapter focuses on the methodologies of these two vaccine strategies: I. purification of hsp110 and grp170 from tumor tissue or cell lines; II. generation and characterization of in vitro HSP-antigen complexes by heat shock using recombinant HSPs derived from a baculovirus protein expression system.     

Live antigen carriers as tools for improved anti-tuberculosis vaccines

Recombinant (r) Mycobacterium bovis BCG strains have been constructed which secrete biologically active listeriolysin (Hly) fusion protein of Listeria monocytogenes. In human and murine macrophage-like cell lines, intracellular persistence of these r-BCG strains was reduced as compared to the parental BCG strain. By immunogold labelling Hly was detected in membrane structures and within the phagosomal space of macrophages. Hly fusions consistently co-localized with a lysosome-associated membrane glycoprotein (LAMP-1) suggesting that membrane attack conformation of Hly was not altered. Although r-BCG microorganisms apparently did not egress into the cytoplasmic compartment of host cells, they both improved major histocompatibility complex class I presentation of co-phagocytosed soluble ovalbumin as compared with wild-type BCG microbes. These data suggest that Hly secretion endows BCG with an improved capacity to stimulate CD8 T cells. Because CD8 T cells play a major role in protection against tuberculosis such Hly-secreting r-BCG constructs are anti-tuberculosis vaccine candidates. In addition, we report on our r-Salmonella typhimurium expression system combined with the HlyB/HlyD/ TolC export machinery for delivering the prominent mycobacterial antigen Ag85B for immune recognition.     

LTB-Syn: a recombinant immunogen for the development of plant-made vaccines against synucleinopathies

Planta - A recombinant antigen targeting α-synuclein was produced in the plant cell rendering an immunogenic protein capable to induce humoral responses in mice upon oral administration....