Enhanced humoral response to influenza vaccine in aged mice with a novel adjuvant,rOv-ASP-1

Immunization is the best way to prevent seasonal epidemics and pandemics of influenza. There are two kinds of influenza vaccines available in the United States: an inactivated vaccine (TIV) and an attenuated vaccine; however, only TIV is approved for immunization of the elderly population. While the aged population has the highest rate of influenza vaccination, the protective efficacy is low as evidenced by elderly individuals having the highest mortality associated with influenza. Recently, we reported that an adjuvant derived from the helminth parasite Onchocerca volvulus, named O. volvulus activation-associated secreted protein-1 (Ov-ASP-1), can significantly enhance the protective efficacy of an inactivated vaccine (TIV) in young adult mice. In the current study, we examined whether this recombinant Ov-ASP-1 (rOv-ASP-1) can enhance the efficacy of TIV in aged mice as well. While primary immunization with TIV alone produced only a low level of influenza-specific antibodies (total IgG, IgG1, and IgG2c) in aged mice, the antibody levels were significantly increased after immunization with TIV + rOv-ASP-1. More importantly, the level of the total IgG in aged mice administered TIV + rOv-ASP-1 was comparable to that of young adult mice immunized with TIV alone. Co-administration of rOv-ASP-1 induced a low level of cross-reactive antibody and enhanced the protective efficacy of TIV in aged mice, reflected by significantly increased survival after challenge with a heterologous influenza virus. rOv-ASP-1 was also superior to the conventional adjuvant alum in inducing specific IgG after TIV immunization in aged mice, and in conferring protection after challenge. These results demonstrate that rOv-ASP-1 may serve as a potential adjuvant for influenza vaccine to improve the efficacy of protection in the elderly.     

DNA immunisation with Onchocerca volvulus chitinase induces partial protection against challenge infection with L3 larvae in mice

The parasitic nematode, Onchocerca volvulus is a major cause of blindness and dermal pathology in tropical regions. A vaccine directed to infective larvae would provide a valuable control tool alongside the current methods of chemotherapy and vector control. Previously we have described the identification of a chitinase cDNA that is expressed in a stage specific manner by O. volvulus infective third stage (L3) larvae. To evaluate its host protective potential, the complete open reading frame was cloned into the eukaryotic expression plasmid pJW4303 and used to vaccinate mice by DNA immunisation with the Accell GeneGun. The survival of challenge infective larvae was monitored using implanted micropore chambers. In the first trial, mice immunised 3 times over 4 months with 1 μg O. volvulus chitinase DNA responded with modest antibody responses dominated by IgG2a and exhibited a 36% (p=0.189, NS) reduction in parasite survival compared with challenge controls. In the second trial, an increased dose of DNA (5 μg) and more frequent immunisations (5 times over 6 months) stimulated an IgG1 dominant response and a 53% reduction in parasite survival (p=0.042). Antibodies from the vaccinated mice reacted with the cuticle of post-infective L3 larvae, implying that this may be the site of immune attack following secretion of chitinase.     

A truncated fragment of Ov-ASP-1 consisting of the core pathogenesis-related-1 (PR-1) domain maintains adjuvanticity as the full-length protein

The Onchocerca volvulus activation-associated secreted protein-1 (Ov-ASP-1) has good adjuvanticity for a variety of antigens and vaccines, probably due to its ability activate antigen-processing cells (APCs). However, the functional domain of Ov-ASP-1 as an adjuvant is not clearly defined. Based on the structural prediction of this protein family, we constructed a 16-kDa recombinant protein of Ov-ASP-1 that contains only the core pathogenesis-related-1 (PR-1) domain (residues 10–153), designated ASPPR. We found that ASPPR exhibits adjuvanticity similar to that of the full-length Ov-ASP-1 (residues 10–220) for various antigens, including ovalbumin (OVA), HBsAg protein antigen, and the HIV peptide 5 (Pep5) antigen, but it is more suitable for vaccine design in ASPPR-antigen fusion proteins, and more stable in PBS than Ov-ASP-1 stored at −70 °C. These results suggest that ASPPR might be the functional region of Ov-ASP-1 as an adjuvant, and therefore could be developed as an adjuvant for human use.     

Type 2 immune-inducing helminth vaccination maintains protective efficacy in the setting of repeated parasite exposures

Animal studies have demonstrated that helminth vaccines which induce type 2 immune responses can be protective. To date, however, such vaccines have not been tested against repeated parasite challenges. Since repeated antigenic challenge of patients with allergic disease results in immunologic tolerance, we hypothesized that a helminth vaccine which induces type 2 immune responses may lose its protective efficacy in the setting of repeated parasite exposures (RPEs). To test this hypothesis, we examined whether RPEs induce immunological tolerance and reduce the effectiveness of a type 2 immune-inducing vaccine. BALB/c mice vaccinated against Litomosoides sigmodontis, a filarial nematode of rodents, were repeatedly exposed to irradiated larvae for 2 or 8 weeks or to non-irradiated infectious larvae for three months.     

Further trials of mebendazole and metrifonate in the treatment of onchocerciasis

In clinical drug trials, mebendazole, given in various daily doses for up to 14 days to a maximum total of 16.8 g, was found to be ineffective against both adult Onchocerca volvulus and the microfilariae. Metrifonate, however, given in doses of 100 mg daily for 14 days or as four doses of 10 mg/kg of body weight either on alternate days or at weekly intervals showed moderate activity against the microfilariae but none against the adult worm.     

Recombinant Calponin of human filariid Brugia malayi: Secondary structure and immunoprophylactic potential

In the search for potential vaccine candidates for the control of human lymphatic filariasis, we recently identified calponin-like protein, that regulates actin/myosin interactions, in a proinflammatory fraction F8 (45.24–48.64 kDa) of Brugia malayi adult worms. In the present study, the gene was cloned, expressed, and the recombinant Calponin of B. malayi (r-ClpBm) was prepared and characterized. r-ClpBm bears homology with OV9M of Onchocerca volvulus, a non-lymphatic filariid that causes loss of vision and cutaneous pathology. r-ClpBm was found to be a ∼45 kDa protein that folds into a predominantly α-helix conformation. The protective efficacy of r-ClpBm against B. malayi infection in Mastomys coucha was investigated by assessing the course of microfilaraemia and adult worm burden in the host immunized with r-ClpBm and subsequently infected with infective third stage larvae (L₃). Expression of the Calponin was detected in all life stages (microfilariae, L₃, L₄, L₅ and adults) of the parasite and immunization with r-ClpBm partially protected M. coucha against establishment of infection as inferred by ∼42% inhibition in parasite burden. Upregulated cellular proliferation, TNF-α, IFN-γ, IL-1β, IL-4, nitric oxide (NO) release, expression of iNOS, and specific IgG, IgG1 and IgG2b in immunized animals correlated with parasitological findings. r-ClpBm immunization caused degranulation in majority of mast cells indicating possible involvement of mast cell products in reducing the parasite survival. It appears that complex mechanisms including Th1, Th2, NO and mast cells are involved in the clearance of infection. To the best of our knowledge this is the first report on cloning, expression of the gene and purification of r-ClpBm, determination of its secondary structure and its ability to partially prevent establishment of B. malayi infection. Thus, r-ClpBm may further be studied and developed in combination with other protective molecules of B. malayi as a component of potential filarial cocktail vaccine candidate.     

Experiments on the chemoprophylaxis of Onchocerca volvulus infection

Previous work on monkeys and on human volunteers led to the development of a schedule of diethylcarbamazine dosage suitable for the chemoprophylaxis of loiasis. In several parts of Africa where this chemoprophylaxis is practised against Loa loa, infections with Onchocerca volvulus are also common. Attempts were therefore made to determine whether diethylcarbamazine has any prophylactic action against the latter parasite by making use of chimpanzees exposed to experimental infections, and also by using biopsy techniques to study the fate of infective larvae inoculated into volunteers.     

The effects of drugs on Onchocerca volvulus. 4. Trials of melarsonyl potassium

The effects of the arsenical drug melarsonyl potassium on Onchocerca volvulus were investigated in patients in Cameroon infected with the Cameroon forest and Sudan savanna strains of the parasite. Two intramuscular dosage schedules were tested: the first comprised 4 consecutive daily doses of 200 mg repeated once after a 10-14 day interval, i.e., 2 (4×200 mg). The second was a single dose schedule at 7.1 mg/kg—10 mg/kg, with a maximum of 500 mg.     

Histochemical enzyme variation in Onchocerca volvulus microfilariae from rain-forest and Sudan-savanna areas of the Onchocerciasis Control Programme in West Africa

Histochemical staining methods for acid phosphatase were used to study the differences among microfilariae of various West African strains of Onchocerca volvulus in both forest and Sudan-savanna onchocerciasis zones. The results have shown statistically significant differences in the staining patterns of microfilarial populations in the two zones. In the rain-forest areas, where onchocerciasis is transmitted by Simulium yahense, S. sanctipauli, S. soubrense and S. squamosum, there were no significant differences of microfilarial staining patterns in patients, by age and sex, between the three Simulium—Onchocerca complexes studied. There was a close relationship between the “strain differences”, as revealed morphoenzymatically, and the clinical picture of the disease in both the forest and the Sudan-savanna zones. The present findings are in favour of the hypothesis that there are intrinsic differences in the strains of the parasite occurring in the two areas. The application of the histochemical means of parasite characterization appears to be a useful tool in differentiating strains of O. volvulus and could contribute towards a better understanding of the epidemiology of human onchocerciasis in different bioclimatic zones where the disease is endemic.     

Onchocerciasis in Zambia: report of O. volvulus in a child and its differentiation from O. dukei in cattle

We report the finding of Onchocerca volvulus in a child in Zambia. This is the first such documented case in an indigenous person and represents perhaps the southernmost limit of the known occurrence of human onchocerciasis in Africa. The specific diagnosis was confirmed by the morphology of the adult worms and microfilariae in tissue sections. O. volvulus was differentiated from O. dukei, a similar species found in cattle from the same geographical locality, to exclude a zoonosis.     

Onchocerca volvulus: comparison of field collection methods for the preservation of parasite and vector samples for PCR analysis.

In recent years, methods for the identification of the filarial worm Onchocerca volvulus and its vector, blackflies of the Simulium damnosum complex (S. damnosum sensu lato (s.l.)), based on the amplification of parasite and vector DNA sequences with the polymerase chain reaction (PCR), have been developed. Routine application of these methods requires techniques for sample collection and preservation that are compatible with the limitations of field collection, yet preserve DNA in a form suitable for PCR. Two different methods for sample preservation were evaluated by the field collection teams and the DNA probe laboratory of the Onchocerciasis Control Programme in West Africa. The most successful involved the preservation of material from O. volvulus and its associated vectors in a dried state on microscope slides. Of over 1200 parasite samples preserved in this manner, more than 93% retained DNA yielding positive results in PCR analysis (1208/1291). Vector material (malpighian tubules and ovaries) preserved in the same manner on the same microscope slides also yielded DNA that was suitable for PCR.     

Induction of protection against divergent H5N1 influenza viruses using a recombinant fusion protein linking influenza M2e to Onchocerca volvulus activation associated protein-1 (ASP-1) adjuvant

Our previous studies have shown the adjuvanticity of an Onchocerca volvulus recombinant protein, Ov-ASP-1 (ASP-1), when administered in an aqueous formulation with bystander vaccine antigens or commercial vaccines. In this study, we reported a novel formulation that took advantage of the protein nature of the ASP-1 adjuvant by creating recombinant fusion protein vaccines linking the highly conserved extracellular domain of M2 protein (M2e) consensus sequence of H5N1 influenza viruses with the ASP-1 adjuvant. Two recombinant fusion proteins designated M2e-ASP-1 and M2e3-ASP-1 were studied, in which ASP-1 was fused with one or three tandem copies of the M2e antigen. Our results show that these novel recombinant influenza vaccines, particularly M2e3-ASP-1, induced strong anti-M2e-specific humoral and cellular immune responses in the established mouse model. Furthermore, M2e3-ASP-1 was able to provide significant cross-clade protection against divergent H5N1 viruses. Consequently, this study has demonstrated a potential novel vaccine formulation that could provide a complementary prophylactic strategy in preventing the threat of future influenza outbreak resulting from rapid evolution of the H5N1 virus and co-circulation of multiple antigenic variants in various regions.     

The intake and transmissibility of Onchocerca volvulus microfilariae by Simulium damnosum fed on patients treated with Diethylcarbamazine, suramin or Mel W

The best hope for the control of transmission of Onchocerca volvulus over much of Africa lies in combining larvicidal measures directed against the Simulium vectors with chemotherapeutic measures designed to reduce the reservoir of transmissible microfilariae in man.     

Ocular onchocerciasis.

Well over 20 million people in the world are infected with Onchocerca volvulus and it is probable that 200 000-500 000 people are blind as a result of this infection, which is the most important cause of blindness in certain areas of Africa and Latin America.     

Inflammatory reactions in onchocerciasis: a report on current knowledge and recommendations for further study.

This report concerns the host''s reactions to the presence of the parasite both in the course of the natural disease and during drug treatment. The various stages of Onchocerca volvulus are discussed in terms of the type of tissue reaction seen. The discussion then turns to basic hypotheses concerning the etiology of these reactions, emphasis being placed on the fact that while pathological changes are considerable in some locations there is a remarkable lack of reaction in others. Some of the mechanisms possibly involved in this apparent absence of host response are discussed, including anti-complement factors, poor antigenicity, acquisition of host antigen, immune tolerance, and blocking antibodies. In any study of the inflammatory response it is recommended that critical evaluations be made of histological material, haematological studies, the definition of the antigenic nature of O. volvulus, characterization of immunological reactivity of patients, and the definition of the migratory pathways of the parasite.     

Glyceradehyde-3-phosphate dehydrogenase as a suitable vaccine candidate for protection against bacterial and parasitic diseases

The enzyme glyceraldehyde-3-P-dehydrogenase (GAPDH) has been identified as having other properties in addition to its key role in glycolysis. The ability of GAPDH to bind to numerous extracellular matrices, modulation of host-immune responses, a role in virulence and surface location has prompted numerous investigators to postulate that GAPDH may be a good vaccine candidate for protection against numerous pathogens. Although immune responses against GAPDH have been described for many microorganisms, vaccines containing GAPDH have been successfully tested in few cases including those against the trematode—Schistosoma mansoni, the helminth—Enchinococcus multilocularis; the nematode filaria— Litomosoides sigmodontis; fish pathogens such as Aeromonas spp., Vibrio spp., Edwarsiella spp., and Streptococcus iniae; and environmental streptococci, namely, Streptococcus uberis and Streptococcus dysgalactiae. Before GAPDH-based vaccines are considered viable options for protection against numerous pathogens, we need to take into account the homology between the host and pathogen GAPDH proteins to prevent potential autoimmune reactions, thus protective GAPDH epitopes unique to the pathogen protein must be identified.     

Simulium species of the amazonicum group as vectors of Mansonella ozzardi in the Brazilian Amazon

Experimental infection with Mansonella ozzardi of common haematophagous Diptera collected at a Ticuna Indian village on the upper reaches of the Solimões river in the Brazilian Amazon, showed that Simulium amazonicum and Simulium n.sp. are capable of supporting full development of the parasite. Natural infections with this filaria were found in both species including infective larvae in Simulium n.sp. No development of M. ozzardi occurred in Mansonia amazonensis, Culicoides insinuatus or Lepiselaga crassipes (Tabanidae). The dimensions of developing larvae of M. ozzardi in both species of black-fly were recorded. Infective larvae of this species may easily be distinguished from those of Onchocerca volvulus, also transmitted in the Amazon by a species closely resembling S. amazonicum, by the presence of a bifid tail and higher anal ratio in M. ozzardi.     

The effects of drugs on Onchocerca volvulus. 2. The antimonial preparations TWSb and MSbE

Antimonial preparations (Pentostam, Neostibosan, stibophen, and tartar emetic) have occasionally been used in the treatment of onchocerciasis without very promising results. The advent of the preparations TWSb (stibocaptate) and MSbE (Friedheim) of allegedly reduced toxicity made it desirable to test them against Onchocerca volvulus.     

Evaluation of PCR-based methods for the diagnosis of infection in bancroftian filariasis

The value of the polymerase chain reaction (PCR) in the diagnosis of Wuchereria bancrofti infection was evaluated in comparison to microscopical examination of night blood smears, Nuclepore filtration, serology and ultrasonography. No correlation was found between PCR-based deoxyribonucleic acid (DNA) probing and serology. We did not find any evidence of free filarial DNA in either blood plasma or chylocoele fluid. We conclude that the 2 PCR-based techniques evaluated are not more sensitive than Nuclepore filtration for detection of W. bancrofti microfilaraemia, need at least 1 intact microfilaria in the volume of blood used for DNA extraction, and were much inferior to ultrasonography for detection of amicrofilaraemic adult worm carriers.     

Antigenemia in young children living in Wuchereria bancrofti-endemic areas of Orissa, India

The prevalence of filarial antigenemia (an indicator of adult worm burden) among 610 children, aged 3-15 years, was determined in three endemic villages of Khurda District, Orissa, India, during 2005. Prevalence of antigenemia, detected using Og4C3 circulating filarial antigen ELISA, was 32.6% compared with 10% microfilaraemia. Although the prevalence of antigenemia increased marginally with increase in age, no significant difference was observed among the children of different age groups (28.3% in 3-5 years, 31.5% in 6-10 years and 35.2% in 11-15 years), indicating that the adult worm burdens did not vary much according to the age of the study children. Gender did not influence the prevalence of antigenemia. The study emphasizes the advantage of using the circulating filarial antigen assay for detecting true filarial infection and demonstrates a high prevalence of antigenemia among the 610 children studied.