Bernard C. Zook

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.

Different Strains of Mycobacterium tuberculosis Cause Various Spectrums of Disease in the Rabbit Model of Tuberculosis

ABSTRACT The rabbit model of tuberculosis has been used historically to differentiate between Mycobacterium tuberculosis and Mycobacterium bovis based on their relative virulence in this animal host. M. tuberculosis infection in market rabbits is cleared over time, whereas infection with M. bovis results in chronic, progressive, cavitary disease leading to death. Because of the innate resistance of commercial rabbits to M. tuberculosis, 320 to 1,890 log-phase, actively growing inhaled bacilli were required to form one grossly visible pulmonary tubercle at 5 weeks. The range of inhaled doses required to make one tubercle allows us to determine the relative pathogenicities of different strains. Fewer inhaled organisms of the M. tuberculosis Erdman strain were required than of M. tuberculosis H37Rv to produce a visible lesion at 5 weeks. Furthermore, with the Erdman strain, only 7 of 15 rabbits had healed lesions at 16 to 18 weeks; among the other animals, two had chronic, progressive cavitary disease, a phenotype usually seen only with M. bovis infection. Genotypic investigation of the Erdman strain with an H37Rv-based microarray identified gene differences in the RD6 region. Southern blot and PCR structural genetic analysis showed significant differences between M. tuberculosis strains in this region. Correlation of the relative pathogenicity, including disease severity, in the rabbit model with the strain genotype may help identify stage-specific M. tuberculosis genes important in human disease.

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.

The Effects of 860 MHz Radiofrequency Radiation on the Induction or Promotion of Brain Tumors and Other Neoplasms in Rats

Abstract Zook, B. C. and Simmens, S. J. The Effects of 860 MHz Radiofrequency Radiation on the Induction or Promotion of Brain Tumors and Other Neoplasms in Rats. Sprague-Dawley rats were irradiated with a continuous- wave (CW) or a pulsed-wave (P) radiofrequency (RF) for 6 h/day, 5 days/week from 2 up to 24 months of age. The RFs emanated from dipole antennas (1 W average output) 2.0 ± 0.5 cm from the tip of each rats nose. The RFs had an 860 MHz frequency, and the specific absorption rate was 1.0 W/ kg averaged over the brain. Fifteen groups of 60 rats (900 total) were formed from offspring of females injected i.v. with 0 (groups 1, 2, 9, 10, 13), 2.5 (groups 5, 6, 7, 8, 11, 12, 14) or 10 mg/kg (groups 3, 4, 15) ethylnitrosourea (ENU) to induce brain tumors. Groups 1, 3, 5 and 7 received the PRF, and groups 9 and 11 the CWRF; groups 2, 4, 6, 8, 10 and 12 were sham-irradiated, and groups 13–15 were cage controls. All rats but 2, totaling 898, were necropsied, and major tissues were studied histopathologically. There was no statistically significant evidence that the PRF or CWRF induced neoplasia in any tissues. Additionally, there was no significant evidence of promotion of cranial or spinal nerve or spinal cord tumors. The PRF or CWRF had no statistically significant effect on the number, volume, location, multiplicity, histological type, malignancy or fatality of brain tumors. There was a trend for the group that received a high dose of ENU and was exposed to the PRF to develop fatal brain tumors at a higher rate than its sham group; however, the result was not significant using the log-rank test (P = 0.14, 2-tailed). No statistically significant differences were related to the PRF or CWRF compared to controls in the low- or zero-dose groups regarding tumors of any kind.

Susceptibility to Tuberculosis: Clues from Studies with Inbred and Outbred New Zealand White Rabbits

ABSTRACT The rabbit model of tuberculosis (TB) is important because rabbits develop a disease that is similar to TB in humans, namely, granulomas with caseous necrosis, liquefaction, and cavities. We describe here a comparison of inbred and outbred New Zealand White rabbits infected by aerosol with either Mycobacterium tuberculosis Erdman or H37Rv strain. Five weeks after infection with either bacillary strain, the inbred rabbits had significantly larger pulmonary tubercles than did outbred rabbits (2.7 versus 1.4 mm in diameter; P < 0.01). After infection with H37Rv, the inbred rabbits had significantly more pulmonary tubercles than did the outbred rabbits (98 ± 12 versus 33 ± 13; P < 0.01), with more mycobacterial CFU per tubercle (809 ± 210 versus 215 ± 115; P = 0.027) (means ± standard errors of the means). Compared with histologic examination of lung granulomas from outbred rabbits, histologic examination of those from inbred rabbits showed more caseous necrosis, more visible bacilli, and fewer mature epithelioid cells. The delayed-type hypersensitivity (DTH) responses to intradermal tuberculin were significantly lower, and peritoneal macrophages from uninfected inbred rabbits produced significantly less tumor necrosis factor alpha after lipopolysaccharide (LPS) stimulation in vitro than those from the outbred rabbits (2,413 ± 1,154 versus 8,879 ± 966 pg/ml). We conclude that these inbred rabbits were more susceptible to TB than their outbred counterparts and had an impaired ability to contain disease, resulting in more grossly visible tubercles that were larger than those observed in outbred rabbits. Preliminary evidence is presented for a cell-mediated immune defect with lower DTH responses and macrophages that have a decreased ability to respond to in vitro stimulation with LPS or M. tuberculosis infection.

Effect of Vaccination with a Recombinant Fusion Protein Encoding an Astacinlike Metalloprotease (MTP-1) Secreted by Host-Stimulated Ancylostoma caninum Third-Stage Infective Larvae

Laboratory dogs were vaccinated intramuscularly with a recombinant fusion protein (expressed and isolated from Escherichia coli) formulated with the Glaxo SmithKline Adjuvant System 02 (AS02). The fusion protein encoded Ac-MTP-1, a developmentally regulated astacinlike metalloprotease secreted by host-stimulated Ancylostoma caninum third-stage larvae (L3). Control dogs were injected intramuscularly with an equivalent amount of AS02 adjuvant alone. The vaccinated and control dogs were then challenged by s.c. injection of 500 L3 of the canine hookworm A. caninum. The vaccinated dogs developed prechallenge immunoglobulin G2 (IgG2) antibody responses specific to anti–Ac-MTP-1-fusion protein with titers ranging between 1:40,000 and 1:364,000, whereas they developed antigen-specific immunoglobulin E antibody responses with titers ranging between 1:500 and 1:1,500. By immunoblotting, canine sera obtained from the vaccinated dogs recognized a protein of the estimated apparent molecular weight of Ac-MTP-1 in activated L3 secretory products. Spearman rank order correlations between the canine intestinal adult hookworm burden and quantitative egg counts at necropsy and anti-Ac-MTP-1 IgG2 antibody titers revealed a statistically significant inverse association (r = −0.89; P = 0.04), suggesting that this molecule offers promise as a recombinant vaccine.

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.

Evaluation of ENU-Induced Gliomas in Rats: Nomenclature, Immunochemistry, and Malignancy

Rats developed mixed gliomas, oligodendrogliomas, and a few astrocytomas in response to transplacental ethylnitrosourea. The neoplastic cell composition of mixed gliomas must be defined; this study required a 20-80% admixture of neoplastic astrocytes and oligodendroglia for the diagnosis of mixed glioma. A battery of immunoantibodies, including Leu-7, S-100, and vimentin, were helpful in classifying rat gliomas, and the histologic features of each tumor type are described. Other brain tumor characteristics that may decide the outcome of carcinogenicity studies include incidence, multiplicity, latency, fatality, size, and malignancy. The size of tumors was determined by measuring their 3-dimensional volumes. Brain tumor volume was found to be highly correlated with malignancy and fatality. Systematic evaluation of the malignancy of brain tumors is an important but often overlooked adjunct method of measuring the effectiveness of a carcinogen. A system to estimate malignancy, one that grades 9 tumor characteristics and weights, each according to clinical outcome, was developed. It was found that mixed gliomas grew larger, had a shorter latency, and were significantly more malignant than were other gliomas.

Preclinical and clinical studies on immunogenicity and safety of the HIV-1 p17-based synthetic peptide AIDS vaccine--HGP-30-KLH.

Immunization with a synthetic HIV-1 p17 peptide analog (HGP-30; aa 85-115 of HIV p17), coupled to a carrier protein (KLH, keyhole limpet hemocyanin) given with alum as the adjuvant induces antibodies which cross-react with both HGP-30 and HIV p17 and clones of cytotoxic and helper T-cells which recognize HGP-30 and HIV p17. Proliferation of lymphocytes in response to HGP-30 has been observed in mice, in HIV-infected individuals and in healthy HIV-seronegative volunteers vaccinated with the p17-based synthetic peptide construct. Cytotoxic T-cell responses against EBV transformed, recombinant p17 pulsed targets were observed using antigen-expanded PBLs from HGP-30-KLH immunized individuals. These results are consistent with predictions that the HGP-30 domain of HIV p17 contains both T- and B-cell epitopes that are recognized by animals and humans. In preclinical toxicology studies in animals and in initial clinical trials in humans the synthetic peptide construct (HGP-30-KLH/alum) has been shown to be safe. This paper summarizes the preclinical immunogenicity and safety data for HGP-30-KLH and presents the initial results from the first Phase 1 clinical trial.

The Effects of Pulsed 860 MHz Radiofrequency Radiation on the Promotion of Neurogenic Tumors in Rats

Abstract Zook, B. C. and Simmens, S. J. The Effects of Pulsed 860 MHz Radiofrequency Radiation on the Promotion of Neurogenic Tumors in Rats. Radiat. Res. 165, 608–615 (2006). In a previous study, this laboratory reported a statistically nonsignificant trend for shortened latency of ethylnitrosourea (ENU)-induced brain tumors in Sprague-Dawley rats exposed to an 860 MHz pulsed radiofrequency (RF) signal. The present study was designed to investigate further any promoting effect of the pulsed RF signal on latency and other characteristics of neurogenic tumors in the progeny of pregnant rats treated with 6.25 or 10 mg/kg ENU. The resulting 1080 offspring were randomized equally by number, sex and ENU dose into pulsed RF, sham and cage control groups. The rats were exposed to the pulsed RF signal 6 h per day 5 days per week; the sham-exposed group was similarly confined for the same periods, and the cage controls were housed in standard cages. An essentially equal number of rats from each group were killed humanely every 30 days between the ages of 171 and 325 days; 32 rats died and 225 rats were killed when they were moribund. Postmortem examinations on the 1080 rats revealed 38 spinal cord tumors, 191 spinal nerve tumors, 232 cranial nerve tumors, and 823 brain tumors. A methodical study of the tumor characteristics disclosed no evidence that exposure to the pulsed RF signal affected the incidence, malignancy, volume, multiplicity, latency or fatality associated with any kind of neurogenic tumor.