Progress on developing a recombinant coccidiosis vaccine

The past 10 years of research aimed at developing subunit vaccines against a number of apicomplexans, including Eimeria, Plasmodium and Toxoplasma, have, if anything, revealed the complex nature of parasite-host interactions. The Knowledge gained from this research has shown why developing a subunit vaccine based on a single recombinant antigen from one developmental stage of the parasite was an overly optimistic approach. Many apicomplexan parasites have acquired unique strategies to evade host immunity. The variable expression of genes encoding erythrocyte membrane protein 1 of Plasmodium falciparum [1] (Berendt et al. Parasitology 1994;108:S19-S28) exemplifies one such strategy. The particular mechanism for evading immune destruction depends on a number of interrelated factors, not least of which is the parasite life-cycle and the availability of susceptible hosts. The goal of any vaccine, be it an attenuated organism or a recombinant antigen, is to break the cycle of infection. The development of a recombinant vaccine against apicomplexan parasites will depend on identifying those antigens and intracellular processes that are vital to the parasite survival and those which exist merely as a way of evading immunity. The information that follows is a review of both molecular biology/biochemistry of eimerian parasites and factors that influence host immune responses to coccidia.     

Enhancing immune responses using suicidal DNA vaccines

We describe a DNA vaccine strategy that allows antigens to be produced in vivo in the context of an alphaviral replicon. Mice immunized with such vectors developed humoral and cellular immune responses at higher levels than mice that received a conventional DNA vaccine vector. Immunized animals acquired protective immunity to lethal influenza challenge. Compared with traditional DNA vaccine strategies in which vectors are persistent and the expression constitutive, the expression mediated by the alphaviral vector was transient and lytic. As a result, biosafety risks such as chromosomal integration, and the induction of immunological tolerance, could be circumvented.     

Novel vaccines for ectoparasites

Novel vaccines against ectoparasites have the potential to be cost-effective new technology for pest control that avoids some of the real and perceived problems with insecticide and acaricide usage. Nevertheless, their development is in its infancy. A vaccine against the cattle tick Boophilus microplus, the world's first vaccine against an ectoparasite, is in field use in Australia. Considerable effort had gone into the development of a vaccine against the sheep blowfly Lucilia cuprina, while other vaccines are at an earlier stage of development. The identification of critical antigens and their production as effective recombinant proteins remains the greatest hurdle. Characteristics of the few known antigens and the mode of action of the protective immune response are discussed. Development of further vaccines will depend on recognition of likely antigenic targets. The efficacy of such vaccines will depend on the characteristics of the target species, in particular its digestive biology and the way in which the novel vaccine impacts on the parasite population.     

HIV gp120-specific cell-mediated immune responses in mice after oral immunization with recombinant Salmonella

Salmonella is of great interest as a potential human immunodeficiency virus vaccine vector because of its ability to elicit potent mucosal and systemic immune responses when administered orally. To determine whether such a vaccine could elicit an immune response in mice, plasmids expressing HIV gp120-LAI were introduced into attenuated S. typhimurium. Three serial doses of 10(10) recombinant organisms were administered orally to BALB/c mice at 2-week intervals. Immunized mice but not control mice demonstrated proliferative T cell responses to gp120-LAI, comparable in magnitude to the proliferative responses to Salmonella antigens. Immunized mice had detectable serum and intestinal Salmonella-specific IgA and serum Salmonella-specific IgG. However, no gp120-specific antibody was detected in either serum or intestinal washes. These results indicate that live recombinant Salmonella-based vaccine constructs can induce HIV-specific cellular immune responses in vivo.     

Serological analysis of human tumor antigens: molecular definition and implications

Specific vaccines for the immunotherapy of human neoplasms require specific human tumor antigens. While efforts to identify such antigens by the analysis of the T-cell repertoire have yielded few antigens, the application of SEREX, the serological identification of antigens by recombinant expression cloning, has brought a cornucopia of new antigens. Several specific antigens have been identified in each tumor tested, suggesting that many human tumors elicit multiple immune responses in the autologous host. The frequency of human tumor antigens, which can be readily defined at the molecular level, facilitates the identification of T-cell-dependent antigens and provides a basis for peptide and gene-therapeutic vaccine strategies.     

Malaria vaccine: roadblocks and possible solutions

Malaria remains the most prevalent and devastating parasitic disease worldwide. Vaccination is considered to be an approach that will complement other strategies for prevention and control of the disease in the future. In the last 10 years, intense studies aimed at the development of a malaria vaccine have provided important knowledge of the nature of the host immunological mechanisms of protection and their respective target antigens. It became well established that protective immune responses can be generated against the distinct stages of Plasmodium. However, in general, protective immune responses are directed at stage-specific antigens. The elucidation of the primary structure of these antigens made possible the generation of synthetic and recombinant proteins that are being extensively used in experimental immunizations against the infection. Today, several epitopes of limited polymorphism have been described and protective immunity can be generated by immunization with them. These epitopes are being tested as primary candidates for a subunit vaccine against malaria. Here we critically review the major roadblocks for the development of a malaria vaccine and provide some insight on how these problems are being solved.     

Stenting of a renal artery bifurcation stenosis

Gene therapy, in particular the transfer of genes encoding immunostimulatory molecules (cytokines and costimulatory molecules) as well as selectively cytotoxic enzymes and DNA vaccination, has the potential of enhancing cell mediated immune responses against tumours including those of colorectal origin. Genes can be transferred using viral vectors either to cultured tumour cells in vitro that can be returned to the patient as a "cancer vaccine", or directly to tumour cells in vivo. Vaccination with DNA constructs expressing specific tumour antigens characteristic of colorectal neoplasia can trigger immune recognition and destruction of tumour cells. The aim is to tip the balance from protumour to antitumour mechanisms by generating a local immune response and systemic antitumour immune memory to destroy metastases. Studies in murine models, combined with human studies, show that such approaches could become an adjunct to current treatments for human colorectal cancer in the near future.     

Conservation of the sporozoite p67 vaccine antigen in cattle-derived Theileria parva stocks with different cross-immunity profiles

Immunity to Theileria parva infection in cattle is often parasite stock specific. The antigenic diversity which is expressed at the schizont stage of the parasite together with a wild reservoir of the organism in buffalo has complicated the development of effective disease control by immunization. We have previously shown that about 70% of cattle inoculated with recombinant forms of p67, a sporozoite stage-specific surface antigen from the cattle-derived Muguga stock of the parasite, are immune to a homologous challenge. Thus, immune responses to p67 can play a role in immunity. The genes encoding this protein in five other parasite stocks have been sequenced. Here, we report that the p67 molecule encoded by four cattle-derived parasite stocks (Boleni, Uganda, Mariakani, and Marikebuni) that fall into different cross-immunity groups is identical in sequence to Muguga p67. The protein encoded by a buffalo-derived parasite exhibits 95% sequence identity with Muguga p67, the major difference being the presence of a 43-residue peptide insert. As predicted by these data, cattle inoculated with recombinant p67 can resist a heterologous cattle-derived parasite challenge. Seven of 12 cattle receiving a homologous Muguga challenge and 6 of 11 cattle receiving a heterologous Marikebuni challenge were immune to East Coast fever. These results extend earlier data suggesting that p67 is a conserved molecule and confirm its potential as a broad-spectrum vaccine antigen for the control of T. parva infection.     

HIV preventive vaccines. Progress to date

The major conceptual problem for HIV vaccine development has been the lack of information on immune responses known to correlate with protection against HIV infection in humans. In this regard, studies on the natural history of HIV infection and AIDS, especially of people with apparent resistance to HIV infection and of patients with HIV infection who have long term survival without disease progression, may provide important information for vaccine development. In addition, a major concern for the development of broadly effective vaccines has been the extensive genetic variability which is characteristic of HIV. In spite of these unknowns, the first generation of HIV candidate vaccines has been developed and evaluated. HIV candidate vaccines based on the subunit recombinant envelope concept (gp120 or gp160) have been shown to protect chimpanzees from HIV infection on challenge, and have now been evaluated in humans in phase I and phase II trials. These products are well tolerated, and capable of inducing neutralising antibodies, but not cytotoxic T lymphocytes. A second vaccine concept, currently in phase I trials, is based on live recombinant vectors, especially using poxvirus vectors followed by boosting with subunit recombinant envelope vaccines. This concept is theoretically very attractive because preliminary data suggest that these vaccines induce both humoral and cell-mediated immunity. However, no published information is available on the ability of live recombinant vector vaccines to protect chimpanzees from HIV infection. The next step in HIV vaccine development is to proceed carefully to expanded phase II and phase III trials to assess the protective efficacy of these candidate vaccines in humans. These trials will be extremely complex from the logistical, scientific and ethical points of view, and will require close collaboration between clinical, basic science and behavioural researchers, national and international organisations, and the pharmaceutical industry.     

Adjuvant and immunostimulating properties of the recombinant Bm86 protein expressed in Pichia pastoris

The cattle tick Boophilus microplus has remained a latent problem to the cattle industry. The recombinant vaccine GAVAC against the cattle tick has proved its efficacy and, conveniently, combined with the use of chemicals could be the solution to this problem. As this vaccine is based in the recombinant concealed antigen Bm86, it has to be given periodically to the animal to maintain an adequate level of antibodies. Some other commercially available vaccines for cattle also have to be given periodically, which creates the possibility of combining vaccines for cattle. In an attempt to evaluate the possible interactions of the Bm86 with other vaccine antigens, a potent stimulatory effect was demonstrated of the recombinant Bm86 on the humoral immune response to the recombinant Hepatitis B surface antigen in mice, and to the inactivated Infectious Bovine Rhinothraqueitis virus in cattle. These results make the Bm86 antigen expressed in Pichia pastoris a good candidate for combining vaccines for cattle because of its dual role, immunogen and adjuvant.     

A simplified vaccinologists' vaccinology and the pursuit of a vaccine against AIDS

Vaccinology is the science and engineering of developing vaccines to prevent infectious diseases. Guidelines come from knowledge of pathogenesis and from successful past vaccines. The vaccine enterprise relies on the evolution of appropriate science and technology. Governmental support and industrial participation are key to successful development of new vaccines. A large challenge for vaccinology is a vaccine which protects against AIDS. Though misguided in its first decade, current vaccine research is directed to use of any and all viral antigens and to elicit both cell-mediated and humoral immune responses that are resident, with memory, at the mucosal sites of viral entry. Recent seminal discoveries guiding the future include selective elicitation of both Type 1 and Type 2 immune responses, and prime-boosting using recombinant viral or DNA vectors and expressed antigens. Success in vaccinology depends on simplification of the complex and on iterative processes in a well-defined pathway. The present and future of vaccinology are discussed in depth.     

Enhancing efficacy of recombinant anticancer vaccines with prime/boost regimens that use two different vectors

BACKGROUND: The identification of tumor-associated antigens and the cloning of DNA sequences encoding them have enabled the development of anticancer vaccines. Such vaccines target tumors by stimulating an immune response against the antigens. One method of vaccination involves the delivery of antigen-encoding DNA sequences, and a number of recombinant vectors have been used for this purpose. To optimize the efficacy of recombinant vaccines, we compared primary and booster treatment regimens that used a single vector (i.e., homologous boosting) with regimens that used two different vectors (i.e., heterologous boosting). METHODS: Pulmonary tumors (experimental metastases) were induced in BALB/c mice inoculated with CT26.CL25 murine colon carcinoma cells, which express recombinant bacterial beta-galactosidase (the model antigen). Protocols for subsequent vaccination used three vectors that encoded beta-galactosidase--vaccinia (cowpox) virus, fowlpox virus, naked bacterial plasmid DNA. Mouse survival was evaluated in conjunction with antibody and cytotoxic T-lymphocyte responses to beta-galactosidase. RESULTS: Heterologous boosting resulted in significantly longer mouse survival than homologous boosting (all P<.0001, two-sided). Potent antigen-specific cytotoxic T lymphocytes were generated following heterologous boosting with poxvirus vectors. This response was not observed with any of the homologous boosting regimens. Mice primed with recombinant poxvirus vectors generated highly specific antibodies against viral proteins. CONCLUSIONS: The poor efficacy of homologous boosting regimens with viral vectors was probably a consequence of the induction of a strong antiviral antibody response. Heterologous boosting augmented antitumor immunity by generating a strong antigen-specific cytotoxic T-lymphocyte response. These data suggest that heterologous boosting strategies may be useful in increasing the efficacy of recombinant DNA anticancer vaccines that have now entered clinical trials.     

Fasciola gigantica: studies of the tegument as a basis for the developments of immunodiagnosis and vaccine

The tegument of bile-dwelling Fasciola gigantica is the interfacing layer that helps the parasite to maintain its homeostasis, and evade the hostile environment, including the host's immune attacks. The tegument is a syncytial layer about 10 mm thick, that is formed by the fusion of cytoplasmic processes of tegument cells, whose soma lie underneath the two muscle layers. The surface of the tegument is highly folded and invaginated into numerous ridges, pits and spines, which help to increase the surface area of the tegument for the absorption and exchanging of molecules, as well as for attachment. The outer membrane covering the tegument is a trilaminate sheet about 12 nm thick, and coated with a carbohydrate-rich glycocalyx layer that also bears high negative charges. Some host molecules may also be adsorbed onto this layer. These unique characteristics enable the parasite to evade the antibody-dependent cell-mediated cytotoxicity (ADCC) reaction exerted by the host. The outer membrane and glycocalyx is continuously replaced by the reserved membrane synthesized and stored in secretory granules of tegument cells, that are transported via cell processes towards the tegument by microtubules. The basal membrane of the tegument is trilaminate and invaginated to form membrane infoldings with closely aligned mitochondria. The tegument cytoskeleton is composed of a highly cross-linked network of 4-6 nm knobby microtrabecular fibers, bundles of intermediate filaments, microtubules that splay out from the tegument cells' processes. Major proteins of the cytoskeleton are actin, paramyosin and tubulin. The flukes' antigens that can elicit strong immunological responses in animal hosts are synthesized and released mainly from the tegument and the cecum. The majority of antigens derived from the surface membrane and the tegument are of MW 97, 66, 58, 54, 47 and 14 kDa, while those released from the cecum are cysteine proteases of MW 27, 26 kDa. Monoclonal antibodies have been raised against some of these antigens, and have been employed in immunodiagnosis of the infection. From the protection conferred to animal models and the in vitro killing assays of young parasites by specific antibodies, candidate vaccines could be selected from these antigens, such as, an antioxidant enzyme, glutathione-S-transferase, the digestive enzyme cysteine proteases, the surface-tegument proteins, such as fatty acid binding protein (14 kDa), membrane proteins (at 66 kDa), as well as muscle protein paramyosin, and hemoprotein. Ongoing research have been directed at deciphering the genetic codes and the syntheses of some of these antigens by recombinant DNA technology.     

New approaches to the development of virus vaccines for veterinary use

The marked progress in recombinant deoxyribonucleic acid (DNA) technology during the past decade has led to the development of a variety of safe new vaccine vectors which are capable of efficiently expressing foreign immunogens. These have been based on a variety of virus types--poxviruses, herpesviruses and adenoviruses--and have led to the production of many new potential recombinant vaccines. Of these recombinant vaccines, the rabies vaccine, in which the rabies G protein is expressed in a vaccinia vector, has been widely used in the field to prevent the spread of rabies both in Europe and in the United States of America. A recombinant Newcastle disease virus vaccine, using fowlpox virus as the vector to express immunogenic proteins from the Newcastle disease virus, has been licensed as the first commercial recombinant vectored vaccine. Many other recombinant virus vaccines are still at the stage of laboratory or field testing. The most recent breakthrough in vaccinology has been the success with the use of naked DNA as a means of vaccination. This approach has shown great promise in mouse model systems and has now become the most active field in new vaccine development. Molecular redesigning of conventional ribonucleic acid (RNA) viruses to obtain more stable attenuated vaccines was previously possible only for positive-strand RNA viruses, such as poliovirus. However, recent advances in molecular biological techniques have enabled the rescuing of negative-strand viruses from DNA copies of their genomes. This has made it possible to engineer specific changes in the genomes of Rhabdoviridae and Paramyxoviridae, both of which include several viruses of veterinary importance. The authors describe the current progress in the development of vector vaccines, DNA vaccines and vaccines based on engineered positive- and negative-strand RNA virus genomes, with special emphasis on their application to diseases of veterinary importance.     

Host-pathogen interaction in amebiasis and progress in vaccine development

Entamoeba histolytica, the causative organism of invasive intestinal and extraintestinal amebiasis, infects approximately 50 million people each year, causing an estimated 40 to 100 thousand deaths annually. Because amebae only infect humans and some higher non-human primates, an anti-amebic vaccine could theoretically eradicate the organism. Uncontrolled epidemiologic studies indicate that acquired immunity to amebic infection probably occurs and that such a vaccine might be feasible. Application of molecular biologic techniques has led to rapid progress towards understanding how Entamoeba histolytica causes disease, and to the identification of several amebic proteins associated with virulence. These proteins are now being evaluated as potential vaccine components. Parenteral and oral vaccine preparations containing recombinant amebic proteins have been effective in preventing disease in a gerbil model of amebic liver abscess. Although systemic and mucosal cellular and humoral immunity both appear to play a role in protection against Entamoeba histolytica, the relative importance of each in the human immune response remains unknown. No animal model of intestinal amebiasis currently exists, moreover, so it has been impossible to evaluate protection against colonization and colitis. Further investigation of the fundamental mechanisms by which Entamoeba histolytica causes disease and of the human immune response to amebic infection is necessary to assess the true feasibility of an anti-amebic vaccine.     

Assessment of the MDA and MDMA optical isomers in a stimulant-hallucinogen discrimination

The mild fowlpox vaccine, FPV M, widely used in Australia is composed of two predominant genotypes based upon differences identifiable in restriction enzyme analyses of plaque purified derivatives of this vaccine. The differences, where identifiable, were in the end fragments of the genomes. Five field isolates of FPV from chickens in New South Wales showed restriction enzyme profiles closely related to the more virulent (standard) vaccine strain, FPV S. The FPV S strain differs from FPV M in both terminal genome fragments and in the presence of a PstI fragment of approximately 10kb (this fragment was also present in PstI digests of all of the field isolates). Plaque purified derivatives of FPV M showed similar lesion development upon inoculation into the wing web of chickens. The field isolates showed significantly higher virulence in day-old and three-week-old chickens in comparison with FPV M. One field isolate was similar to the FPV S vaccine. Two isolates had slowly developing wing web lesions, caused significant secondary lesions in three-week-old chickens and generalised poxvirus infection when inoculated into day-old chickens. For two isolates, the primary wing web lesion took even longer to develop and resolve although these isolates did not cause generalised poxvirus infection. It was possible to identify four virulence/pathogenicity types amongst these vaccine and field isolates of FPV. These strains may allow the characterisation of FPV encoded virulence factors. The field strains with higher virulence may be suitable as parent strains for the construction of FPV recombinants with enhanced immune responses to co-expressed vaccine antigens when compared with current FPV M strain based recombinants.     

Future vaccines

Powerful genetic and immunological techniques allow the production of new vaccines. Recombinant proteins and synthetic peptides represent new categories of subunit vaccines, illustrated, respectively, by recombinant hepatitis B vaccines and a peptide-derived malaria vaccine. Virus and bacteria can be used as vectors of foreign genes encoding antigens of vaccinal interest, to build-up new forms of live vaccines. One may expect from these new strategies of vaccine production a better control of viral, bacterial and parasitic diseases and a dramatic change in vaccine recommendations for children and adults.     

Development of a malaria vaccine

Development of an effective malaria vaccine poses a major scientific challenge both in the laboratory and in the field. Such a vaccine is necessary because of the massive disease burden of malaria in the developing world, the global spread of drug resistance, and the difficulty of sustainable control of the mosquito vector. Animal models have shown the immunological feasibility of vaccines targeted against different stages of parasite development, and studies in human volunteers have shown that a recombinant protein vaccine can protect against challenge with the homologous strain of parasite. However, both natural and vaccine-induced immunity are hampered by the remarkable capacity of the parasites to vary critical antigenic structures; large field trials of a synthetic peptide vaccine gave equivocal results. In an attempt to overcome the dual difficulty of poor immunogenicity and parasite diversity, much experimental work is now focused on complex antigenic constructs, delivered as DNA vaccines or in live vectors such as vaccinia, with multiple targets at each stage of parasite development.     

Neonatal and early life immune responses to various forms of vaccine antigens qualitatively differ from adult responses: predominance of a Th2-biased pattern which persists after adult boosting

Induction of neonatal immune responses to vaccine antigens is believed to be of limited efficacy because of immune immaturity and particular susceptibility to tolerogenic signals during this period of life. To characterize particular features of neonatal immune responses to vaccine antigens, we assessed the capacity of BALB/c mice at different stages of immunological maturation to respond to a selection of vaccine antigens and presentation systems. Significant B and T cell responses to vaccine antigens (tetanus and measles virus peptides, tetanus toxoid, live viral attenuated measles virus, canarypox recombinant measles vector or bacillus Calmette-Guérin) were obtained as early as the first week of life. However, these neonatal responses differed qualitatively from adult responses by a decreased IgG2a/IgG1 ratio of vaccine-specific antibodies, the secretion of significantly higher interleukin-5 and lower interferon-gamma levels by vaccine-specific T cells and an impaired induction of cytotoxic T cell precursors. This pattern of biased Th2 versus Th1 responses induced upon early exposure to vaccines was not reversed by decreasing the doses of vaccine antigens. It did not disappear with aging and was still reflected in adult responses to booster immunization with the corresponding antigen. Thus, neonatal immunization can induce significant vaccine specific responses with a predominance of a Th2 pattern which can persist in boosted adult mice.     

Mixed population approach for vaccination with live recombinant Salmonella strains

Attenuated strains of enteropathogenic species, such as Salmonella, represent useful carries for the delivery of heterologous recombinant antigens to the immune system. A frequently encountered obstacle, however, is the negative influence of high-level antigen production on the stability of carrier strains and the maintenance of their specific properties concerning tissue colonization and viability during infection. To solve this problem we have established an expression system based on genetic variation. This generates two sub-populations of a recombinant vaccine strain, i.e., one consisting of viable cells which maintain all characteristics of the native carrier strain and generate a second population of cells producing antigen(s) of interest at a very high level. This novel expression system offers unique applications and advantages over common live recombinant vaccine approaches.