Philip K. Russell

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.

The proteins of Japanese encephalitis virus.

Polyacrylamide gel electrophoresis of Japanese encephalitis virus (JEV) grown in both LLC-MK2 and chick embryo cell culture revealed three principal polypeptides with molecular weights of 8,700, 13,500, and 53,000 (V-1, V-2, and V-3, respectively). Infected chick cells that were treated with actinomycin D and cycloheximide contained seven polypeptides not present in uninfected cells. In addition to V-2 and V-3, polypeptides with molecular weights of 10,500, 19,000, 45,000, 71,000, and 93,000 (NV-1 through NV-5) were found; V-1 was not regularly detected. A similar pattern of polypeptides was obtained by radioimmune precipitation of soluble antigens from cytoplasmic extracts of infected, actinomycin-D treated, chick cells. When virions were treated with NP-40, a dense, RNA-rich structure was detected which contained V-2. An extracellular, slowly sedimenting, RNA-poor, hemagglutinating particle with a density comparable to the virion was present in virus preparations from cell culture and contained V-1, V-3, and NV-2.

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.

Japanese encephalitis virus glycoproteins.

Abstract Mature Japanese encephalitis (JE) virus, or N-form virus, contained three structural proteins: V-1, V-2, and V-3. The large membrane protein V-3 was glycosylated, whereas both V-1 (the small membrane protein) and V-2 (the nucleocapsid protein) were not. Intracellular (I-form), immature virions from infected chick embryo cells did not contain V-1 but a larger protein NV-2, which was glycosylated. T-form virions, released by LLC-MK2 cells incubated with tris(hydroxymethyl)amino-methane (Tris), also contained the glycoprotein NV-2 instead of the nonglycosylated and smaller V-1. We therefore concluded that JE contained two structural membrane glycoproteins, at least one of which is modified during morphogenesis. The NV-2 polypeptide was heterogeneous, and slight differences in electrophoretic mobility were detected among the NV-2 polypeptide peaks from glucosamine-labeled I-form and T-form virions, glucosamine-labeled cell extracts, and amino acid-labeled cell extracts. The significance of these differences is not clear, but they may indicate that NV-2 is composed of several proteins of similar molecular weight. By analyzing extracts of infected cells labeled with glucosamine or amino acids, we tentatively classified the intracellular polypeptide NV-3 as a virus-specified nonstructural glycoprotein; this polypeptide may be a proteolytic fragment of V-3. The virus-specified polypeptides NV-5, NV-4, and NV-1 were classified as nonglycosylated, nonstructural proteins.

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.

Protein synthesis in japanese encephalitis virus-infected cells

Abstract We studied the effects of actinomycin D, cycloheximide, and puromycin on virus-specified protein synthesis in Japanese encephalitis (JE) virus-infected chick embryo and LLC-MK2 (rhesus monkey kidney) cells. To prove that the radioactively labeled proteins we had previously identified in infected chick cells were virus-specified, we showed that they electrophoretically comigrated with radioactively labeled proteins from infected, but not from uninfected, LLC-MK2 cells. We found that the maximum value for the ratio of protein synthesis in infected chick cells to uninfected cells occurred when the cells were treated with actinomycin D and were pulse-inhibited with cycloheximide. Alone, actinomycin D treatment decreased the background radioactivity of high molecular weight in electropherograms of infected chick cells and allowed virus-specified proteins to be prominent. Cycloheximide pulse-inhibition of infected, actinomycin D-treated cells decreased total cellular protein synthesis and slightly decreased the background radioactivity in electropherograms without changing the distribution of radioactivity among virus-specified proteins. Neither drug treatment decreased the yield of infectious virus. These results differ in some respects from the related results of Trent and Qureshi (1971) . In contrast to our results with cycloheximide, pulse-inhibition of infected chick embryo cells with puromycin inhibited the synthesis of polypeptides NV-5, NV-4, and NV-1 (virus-specified nonstructural, nonglycosylated proteins) to a greater extent than that of V-3, NV-3, NV-2, and V-2 (virus-specified glycosylated and/or structural proteins). It also generally inhibited the synthesis of large proteins relative to small ones. We then studied the effects of puromycin and cycloheximide in LLC-MK2 cells. In contrast to our results in chick embryo cells, pulse-inhibition of infected LLC-MK2 cells with either drug (in the continuous presence of actinomycin D) did not alter the pattern of virus-specified proteins in electropherograms from that obtained without pulse-inhibition. Treatment with continuous levels of either drug (in the presence of actinomycin D) did, however, alter the protein pattern by differentially inhibiting the synthesis of nonstructural, nonglycosylated proteins. By labeling infected cells with one of eight different amino acids, we were unable to find an unusually enriched (or lowered) amino acid content that was common to all nonstructural, nonglycosylated proteins. A possible explanation for the differential inhibition is that the virus directs the formation of functionally different polyribosomes, messengers, or initiation factors which vary in their susceptibility to low levels of inhibitors of protein synthesis.

Assay of arbovirus neutralizing antibody by micro methods

Abstract Efficient, reproducible and specific neutralizing antibody assays are needed, particularly for use in arboviral studies in areas where antigenic cross reactions make serological interpretation difficult. Accordingly, 2 methods were developed for this purpose. A micrometabolic inhibition (MMI) test with BHK-21 cells in disposable “U”-bottomed microtitre plates was used with Japanese encephalitis, Wesselsbron, Sindbis, chikungunya and Batai viruses. The MMI test was highly specific, of efficiency comparable with the haemagglutination-inhibition (HI) test, and was very sparing of reagents and sera. Although equal to or better in sensitivity than conventional cell culture neutralization tests, it was not as sensitive as HI or plaque-reduction neutralization (PRN) tests. The LLC-MK2 cell microculture PRN test was devised for use with the dengue viruses or for use when a combination of precision of antibody measurement and efficiency of testing was required. The microculture and 1 oz. bottle culture PRN tests were of equal specificity. Although not as precise as the bottle PRN test, the microculture PRN test is suitable for routine serology and has the added advantages that it is more efficient and requires less serum and reagents.

Membrane-bound proteins of Japanese encephalitis virus-infected chick embryo cells.

Abstract The seven Japanese encephalitis virus specific polypeptides found in infected chick embryo cells were all bound to membranes. None were completely released from the membranes by treatment with neutral salt, alkaline salt, or dilute detergent, but two of them were partially released by both the neutral and alkaline salts. The polypeptides were released or attacked by trypsin at unequal rates and in the sequence: NV-5≥ NV-4 > V-3. NV-5 was released as a relatively undegraded soluble polypeptide, NV-4 was extensively degraded, and V-3 was degraded but part of its trypsinderived fragment (TF-2) remained membrane bound. We suggest that the three largest viral polypeptides are bound in such a manner that the larger the polypeptide, the more exposed and superficial it is. Treatment of virions with trypsin produced low molecular weight material and three discrete polypeptide fragments, probably all derived from the large virion envelope protein V-3; two (TF-1 and TF-3) had electrophoretic mobilities similar to the two naturally occurring nonvirion virus-specified polypeptides, NV-1 and NV-3.

Immunopathologic Mechanisms in the Dengue Shock Syndrome

Publisher Summary This chapter describes the immunopathologic mechanisms in the dengue shock syndrome (DSS). Immunopathologic mechanisms usually play a minor role in the pathogenesis of disease due to acute viral infections. The relative timing of virus replication and the immune response is a critical factor in determining the degree of damage done by immunopathologic mechanisms in virus diseases. The viruses associated with DSS include all four serotypes of dengue viruses. The same viruses also cause classic dengue fever, and benign undifferentiated febrile illnesses. The major pathophysiologic abnormality seen in DSS is an acute increase in vascular permeability. In typical cases, after a 3- to 6-day febrile period, clinical shock occurs due to loss of plasma from the intravascular space. The onset of the shock phase is acute, and the hematocrit rises sharply as plasma escapes through the endothelium. The hypovolemic shock can lead to tissue hypoxia, acidosis, hyperkalemia, and death if uncorrected. Replacement of plasma volume with colloid-containing solutions is an effective method of treatment which may be lifesaving.

ECOLOGY OF KEYSTONE VIRUS, A TRANSOVARIALLY MAINTAINED ARBOVIRUS*

Our studies in the Pocomoke Cypress Swamp of Maryland have shown that KEY strain of CE is endemic and is carried by the floodwater mosquito A. atlanticus. The virus is transmitted transstadially in nature, as evidenced by our recovery of virus from larvae and males of this species. Serologic evidence, both here and elsewhere, indicates that vertebrates are infected with KEY, but their role in the transmission cycle remains unknown. We have found several animals, for example, the gray squirrel, that are potential vertebrate reservoirs for the virus. Gray squirrels possess antibodies to KEY in nature, are known to be fed upon by A. atlanticus females, and have been shown to circulate a high-titered viremia after experimental inoculation. Evidence from 1974 collections, however, indicates that A. atlanticus females ingested only a single blood meal during the period when adults were active. We will not be able to assess the relative importance of the vertebrate and mosquito cycles until much more work has been performed on vector-reservoir-virus dynamics.