Kashinath Ghosh

Progress in the development of a recombinant vaccine for human hookworm disease: The Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.

Cloning, Yeast Expression, Isolation, and Vaccine Testing of Recombinant Ancylostoma-Secreted Protein (ASP)-1 and ASP-2 from Ancylostoma ceylanicum

cDNAs encoding 2 Ancylostoma-secreted proteins (ASPs), Ancylostoma ceylanicum (Ay)-ASP-1 and Ay-ASP-2, were cloned from infective third-stage larvae (L3) of the hookworm A. ceylanicum and were expressed as soluble recombinant fusion proteins secreted by the yeast Pichia pastoris. The recombinant fusion proteins were purified, adjuvant formulated, and injected intramuscularly into hamsters. Hamsters vaccinated either by oral vaccination with irradiated L3 (irL3) or by injections of the adjuvants alone served as positive and negative controls, respectively. Anti-ASP-1 and anti-ASP-2 antibody titers exceeded 1 : 100000. Each vaccinated hamster was challenged orally with 100 L3. Two groups of vaccinated hamsters (i.e., those vaccinated with either irL3 or ASP-2 formulated with Quil A) exhibited significant reductions in adult hookworm burdens, compared with control hamsters. The hookworms recovered from the hamsters vaccinated with ASP-2 plus Quil A were reduced in length. Splenomegaly, which was observed in control hamsters, was not seen in hamsters vaccinated with either irL3 or ASP-2 formulated with Quil A. These results indicate that ASP-2 is a promising molecule for the development of a hookworm vaccine.

Experimental approaches to the development of a recombinant hookworm vaccine.

Summary: Hookworm infection is a major parasitic cause of morbidity in the developing nations of the tropics. Development of a genetically engineered vaccine would be a useful tool in the control of this infection in highly endemic areas. Recombinant polypeptides belonging to the Ancylostoma secreted protein (ASP)‐1 family have shown promise for reducing hookworm burdens after larval challenge infections in mice. Typically, these polypeptides are expressed in Escherichia coli and administered as an alum precipitate. Vaccine protection is antibody dependent. It is anticipated that a cocktail of different recombinant hookworm antigens may be required in order to effectively prevent heavy hookworm infections and disease. The progress of this work has been hampered by the absence of both a convenient laboratory animal with which to study hookworm infections resembling human infection, as well as the lack of easy availability of native hookworm antigens. In addition, useful human serologic correlates of antihookworm immunity are still poorly defined.

Antibody-Dependent Reductions in Mouse Hookworm Burden after Vaccination with Ancylostoma caninum Secreted Protein 1

Vaccination of mice with either third-stage Ancylostoma caninum infective hookworm larvae (L3) or alum-precipitated recombinant Ancylostoma secreted protein 1 from A. caninum (Ac-ASP-1) results in protection against hookworm challenge infections. Vaccine protection is manifested by reductions in lung hookworm burdens at 48 h postchallenge. Mice actively immunized 4 times with Ac-ASP-1 also exhibited reductions in hookworm burden in the muscles. Hookworm burden reductions from Ac-ASP-1 immunization were associated with elevations in all immunoglobulin subclasses, with the greatest rise observed in host IgG1 and IgG2b. The addition of a fourth immunization resulted in even higher levels of IgG and IgE. In contrast, L3-vaccinated mice exhibited marked elevations in IgG1 and IgM, including anti-Ac-ASP-1 IgM antibody. Passive immunization with pooled sera from recombinant Ac-ASP-1-vaccinated mice also resulted in lung hookworm burden reductions. It is hypothesized that recombinant Ac-ASP-1 vaccinations elicit antibody that interferes with parasite larval migration.

Hookworm burden reductions in BALB/c mice vaccinated with recombinant Ancylostoma secreted proteins (ASPs) from Ancylostoma duodenale, Ancylostoma caninum and Necator americanus.

Vaccination of mice with alum-precipitated recombinant Ancylostoma secreted protein-1 from the canine hookworm Ancylostoma caninum (Ac-ASP-1) results in protection against A. caninum larval challenge. Vaccine protection is manifested by host reductions in hookworm burden compared to control mice. The goal of this study was to determine whether ASP antigens cloned and expressed from different hookworm species will cross protect against A. caninum larval challenge. Cross-species protection against A. caninum challenge infections was observed with immunizations using recombinant ASP-1 from the human hookworms Ancylostoma duodenale and Necator americanus. However, the degree of protection was proportional to the extent of amino acid sequence homology between the ASP immunogen used for vaccination and the Ac-ASP-1 produced by the challenge larval strain. Vaccine protection was noted to decrease significantly as amino acid sequence homologies diverged 10% or more. It was also determined that Ac-ASP-2, a molecule cloned from A. caninum having 55% amino acid sequence homology to the C-terminus of Ac-ASP-1, did not elicit vaccine protection. These observations were partly reflected in the titer of antibodies that recognize Ac-ASP-1. The studies reported here will help to design immunogenic peptide vaccines based on the sequence divergence of hookworm ASPs.

Molecular approaches to vaccinating against hookworm disease

Anthelminthic drug chemotherapy has failed as an acceptable approach to hookworm control in the less developed countries of the tropics. The development of a genetically engineered vaccine against hookworm infection would be a major advance in our efforts to control this parasitic disease. We have produced several lead recombinant hookworm vaccine antigens. Their development is based on scientific principles that were generated almost 70 years ago when investigators first began to attenuate living infective hookworm larvae. Those early studies on attenuated live vaccines highlighted the importance of secreted larval antigens for eliciting protective immunity in dogs challenged with Ancylostoma caninum. The two major secreted larval antigens have been recently identified as Ancylostoma secreted protein-1 (ASP-1) and ASP-2. The predicted amino acid sequences of the ASP cDNAs together with experimental immunogenicty data using the expressed recombinant protein suggest that the ASPs are promising vaccine antigens. Preliminary hookworm challenge data in mice immunized with recombinant ASP-1 helps to validate this assumption. Alternative vaccines based on either genetic immunization (DNA vaccines) or immunization with recombinant molecules expressed from adult hookworm cDNAs are also under evaluation. Optimization of vaccine route, delivery system, and adjuvant formulations will be required before future planned phase I testing in humans. Vaccine development for a target population living in rural areas of less developed countries will require innovative solutions to financing and manufacture.

Interspecific hybridization and genetic variability of Phlebotomus sandflies

The first successful hybridization is reported between Phlebotomus papatasi and P. duboscqi, two important Old World sandfly vectors of leishmaniasis and other diseases. Laboratory strains of P. papatasi and P. duboscqi were separable by six diagnostic enzyme loci: Est‐3, Idh‐1, Mdh‐2, Mpi, Tre‐1 and Tre‐3. Hybrids between the two species were verified by the recovery of heterozygous isozyme patterns for the diagnostic loci. No F2 or backcross progeny were obtained. P. papatasi was separated from P. bergeroti by three diagnostic enzyme loci: Est‐3, Mpi and Pgd. The isozyme patterns of P. bergeroti contain elements of both P. duboscqi and P. papatasi, although seven diagnostic loci (Est‐3, Idh‐1, Me, Mpi, Pgd, Tre‐1 and Tre‐3) separated P. bergeroti from P. duboscqi. Genetic variability profiles of the three species were established for 20 enzyme loci. Three geographically distant strains of P. papatasi from Calcutta, Maharashtra and Israel had isozyme genetic distances of < 0.05. The recently established Calcutta strain showed an unexpectedly low genetic variability with only one (Idh‐2) of 20 loci being polymorphic (average heterozygosity of 1.9%) in contrast to 5–8 polymorphic loci (10–12% heterozygosity) in the Maharashtra and Israel strains. Mass and single pair crosses between the three P. papatasi strains were fertile with normal progeny numbers. Thus we found no signs of speciation in P. papatasi.

The effect of anti-sandfly saliva antibodies on Phlebotomus argentipes and Leishmania donovani

A study was undertaken to find the effect of repeated bites of the sandfly, Phlebotomus argentipes, on its host as well as on the vector itself. The study also aimed to find the effect of the immune serum on the parasite, Leishmania donovani, naturally transmitted by the vector. The hamster which was exposed to sandfly feeding showed good antibody titre against the sandfly salivary-gland secretion, which indicates that the salivary-gland secretion is immunogenic in nature. The result also revealed that the feeding attraction of the females, which has been expressed as the percentage of engorgement, gradually decreased as the mortality rate increased during the subsequent bites. Similar mortality rate was observed when the flies were fed with the immune sera through an artificial membrane feeding method. When the sandflies were fed both with the immune sera and the blood-parasite (L. donovani) suspension, in addition to the major loss of the number of vectors, there was an inhibition of development in the gut and a concomitant reduction in the migration of the parasite in the surviving females. These results indicate that the anti-sandfly saliva immune sera probably bind with the respective antigen-presenting sites of the sandfly salivary gland and, thus, cause the sandfly death. The possible explanation of the inhibition of the forward movement of the parasites is that the attraction of the parasites to the oesophagus, mediated by the sandfly saliva, is inhibited by the anti-saliva antibodies. The importance of anti-sandfly saliva antibodies as a tool of vector control and also to block the transmission of leishmaniasis has been indicated.

EFFECT OF VACCINATIONS WITH RECOMBINANT FUSION PROTEINS ON ANCYLOSTOMA CANINUM HABITAT SELECTION IN THE CANINE INTESTINE

Laboratory dogs were vaccinated subcutaneously with 3 different recombinant fusion proteins, each precipitated with alum or calcium phosphate. The vaccinated dogs were then challenged orally with 400 third-stage infective larvae (L3) of the canine hookworm, Ancylostoma caninum. The 3 A. caninum antigens selected were Ac-TMP, an adult-specific secreted tissue inhibitor of metalloproteases; Ac-AP, an adult-specific secreted factor Xa serine protease inhibitor anticoagulant; and Ac-ARR-1, a cathepsin D–like aspartic protease. Each of the 3 groups comprised 6 male beagles (8 ± 1 wk of age). A fourth group comprised control dogs injected with alum. All of the dogs vaccinated with Ac-TMP or Ac-APR-1 exhibited a vigorous antigen-specific antibody response, whereas only a single dog vaccinated with Ac-AP developed an antibody response. Dogs with circulating antibody responses exhibited 4.5–18% reduction in the numbers of adult hookworms recovered from the small intestines at necropsy, relative to alum-injected dogs. In contrast, there was a concomitant increase in the number of adult hookworms recovered from the colon. The increase in colonic hookworms was as high as 500%, relative to alum-injected dogs. Female adult hookworms were more likely to migrate into the colon than were males. Anti-enzyme and anti-enzyme inhibitor antibodies correlated with an alteration in adult hookworm habitat selection in the canine gastrointestinal tract.

Naturally Occurring Culturable Aerobic Gut Flora of Adult Phlebotomus papatasi, Vector of Leishmania major in the Old World

Background Cutaneous leishmaniasis is a neglected, vector-borne parasitic disease and is responsible for persistent, often disfiguring lesions and other associated complications. Leishmania, causing zoonotic cutaneous leishmaniasis (ZCL) in the Old World are mainly transmitted by the predominant sand fly vector, Phlebotomus papatasi. To date, there is no efficient control measure or vaccine available for this widespread insect-borne infectious disease. Methodology/Principal Findings A survey was carried out to study the abundance of different natural gut flora in P. papatasi, with the long-term goal of generating a paratransgenic sand fly that can potentially block the development of Leishmania in the sand fly gut, thereby preventing transmission of leishmania in endemic disease foci. Sand flies, in particular, P. papatasi were captured from different habitats of various parts of the world. Gut microbes were cultured and identified using 16S ribosomal DNA analysis and a phylogenetic tree was constructed. We found variation in the species and abundance of gut flora in flies collected from different habitats. However, a few Gram-positive, nonpathogenic bacteria including Bacillus flexus and B. pumilus were common in most of the sites examined. Conclusion/Significance Our results indicate that there is a wide range of variation of aerobic gut flora inhabiting sand fly guts, which possibly reflect the ecological condition of the habitat where the fly breeds. Also, some species of bacteria (B. pumilus, and B. flexus) were found from most of the habitats. Important from an applied perspective of dissemination, our results support a link between oviposition induction and adult gut flora.