Prior exposure to an attenuated Listeria vaccine does not reduce immunogenicity: pre-clinical assessment of the efficacy of a Listeria vaccine in the induction of immune responses against HIV.

BACKGROUND: We have evaluated an attenuated Listeria monocytogenes (Lm) candidate vaccine vector in nonhuman primates using a delivery regimen relying solely on oral vaccination. We sought to determine the impact of prior Lm vector exposure on the development of new immune responses against HIV antigens. FINDINGS: Two groups of rhesus macaques one Lm naive, the other having documented prior Lm vector exposures, were evaluated in response to oral inoculations of the same vector expressing recombinant HIV-1 Gag protein. The efficacy of the Lm vector was determined by ELISA to assess the generation of anti-Listerial antibodies; cellular responses were measured by HIV-Gag specific ELISpot assay. Our results show that prior Lm exposures did not diminish the generation of de novo cellular responses against HIV, as compared to Listeria-naïve monkeys. Moreover, empty vector exposures did not elicit potent antibody responses, consistent with the intracellular nature of Lm. CONCLUSIONS: The present study demonstrates in a pre-clinical vaccine model, that prior oral immunization with an empty Lm vector does not diminish immunogenicity to Lm-expressed HIV genes. This work underscores the need for the continued development of attenuated Lm as an orally deliverable vaccine.

Dose-response model for Listeria monocytogenes-induced stillbirths in nonhuman primates.

A dose-response model using rhesus monkeys as a surrogate for pregnant women indicates that oral exposure to 10(7) CFU of Listeria monocytogenes results in about 50% stillbirths. Ten of 33 pregnant rhesus monkeys exposed orally to a single dose of 10(2) to 10(10) CFU of L. monocytogenes had stillbirths. A log-logistic model predicts a dose affecting 50% of animals at 10(7) CFU, comparable to an estimated 10(6) CFU based on an outbreak among pregnant women but much less than the extrapolated estimate (10(13) CFU) from the FDA-U.S. Department of Agriculture-CDC risk assessment using an exponential curve based on mouse data. Exposure and etiology of the disease are the same in humans and primates but not in mice. This information will aid in risk assessment, assist policy makers, and provide a model for mechanistic studies of L. monocytogenes-induced stillbirths.

Use of flow cytometry in an apoptosis assay to determine pH and temperature stability of shiga-like toxin 1.

Shiga toxins and Shiga-like toxins (Stx) are a relatively large group of cytotoxins produced by certain serotypes of Shigella and E. coli (STEC). These toxins are responsible for diarrhea, hemorrhagic colitis and may induce hemolytic uremic syndrome (HUS) with serious consequences in young children. The toxins are proteins made up of 5 small B subunits responsible for binding to an outer membrane ligand on host cells and surround the larger, biologically active A subunit. For Shiga-like toxin 1 (Stx1), the cellular receptor is the carbohydrate globotriose. Stx1was purified from STEC. We utilized induction of apoptosis in the human monocyte cell line THP-1, as a biological endpoint to test the stability of Stx1 activity added to fruit punch at different pH (2-9) and temperatures (4 and 20 degrees C). A flow cytometric method was used to test for early and late apoptotic events based on binding of R-phycoerytherin-labeled annexin V to exposed membrane phosphatidyl serine. Membrane permeability to 7-Amino-actinomycin corresponds with late apoptosis or necrosis. The combination of acid pH and higher storage temperature resulted in greatest degree of toxin inactivation. This approach provides a rapid and high throughput method to determine the functional activity of Stx1, and related toxins in a food matrix.

A synthetic polypeptide based on human E-cadherin inhibits invasion of human intestinal and liver cell lines by Listeria monocytogenes.

Internalin A is a surface protein of the facultative intracellular pathogen Listeria monocytogenes that interacts with the human host cell protein E-cadherin to facilitate invasion of epithelial cells. A single amino acid substitution at position 16 in mouse E-cadherin prevents this interaction. Synthetic polypeptides of 30 aa encompassing position 16 of human and mouse E-cadherin were tested for their ability to inhibit in vitro invasion of Caco-2, HepG2 and TIB73 cell lines by L. monocytogenes. Only the human-derived peptide was capable of inhibiting invasion in the human-origin Caco-2 and HepG2 cell lines. These findings demonstrate that small polypeptides can inhibit invasion of biologically relevant cell types by L. monocytogenes in vitro and may be potentially useful as therapeutic agents in vivo.

Differential reactive oxygen and nitrogen production and clearance of Salmonella serovars by chicken and mouse macrophages.

The objective of the present study was to compare the uptake and killing of Salmonella serovars by murine and avian macrophage cell lines. We used Salmonella enterica serovars Enteritidis (SE338) and Typhimurium (SR11) for this study. Uptake of green fluorescent protein-labeled bacteria was measured using flow cytometry. Cell sorting and plating of viable infected macrophages demonstrated that bacterial clearance was significantly better with J774A.1 compared with HD11 cells. HD11 cells produced significantly higher amounts of nitric oxide (NO) than J774A.1 cells upon infection with SE338 and SR11, whereas J774A.1 cells exhibited greater superoxide production with SR11. Treatment of HD11 cells with recombinant chicken interferon gamma in the absence of bacteria enhanced NO production but did not induce increased levels synergistically with bacteria. Interferon treatment did not influence phagocytosis or increase killing by HD11 cells.

Differential responses of macrophages to Salmonella enterica serovars Enteritidis and Typhimurium.

Macrophages are major effectors against Salmonella infection, and also transport bacteria between host tissues and provide a protected site for intracellular bacterial replication. We hypothesized that differences in chicken macrophage responses to Salmonella enterica serovar Enteritidis (SE) and serovar Typhimurium (ST) played a role in preferential infection of eggs by SE compared with ST. To test this hypothesis, we determined bacterial phagocytosis and intracellular viability and macrophage nitric oxide (NO) production following in vitro infection with SE or ST in the presence or absence of interferon-gamma (IFN-gamma). The effects of bacterial components, lipopolysaccharide (LPS), outer membrane proteins (OMP) and flagella, on NO production were also assessed. Our results showed: (1) in the presence or absence of IFN-gamma, the percentage macrophages phagocytizing SE and ST was similar; (2) the number of intracellular viable SE was significantly reduced compared with ST in the presence or absence of IFN-gamma; (3) increased macrophage necrosis was seen in the presence of IFN-gamma and ST; (4) Salmonella infection acted synergistically with IFN-gamma in induction of nitric oxide production; and (5) in the absence of IFN-gamma, macrophages produced significantly greater NO following treatment with SE outer membrane protein or flagella compared with ST OMP or flagella, while in the presence of IFN-gamma significantly less NO was produced following treatment with SE-LPS compared with ST-LPS. These results suggest that differential responses of chicken macrophages to SE versus ST may result in increased macrophage death with ST, which could result in an increased inflammatory response as compared to SE.

CpG-induced immunomodulation and intracellular bacterial killing in a chicken macrophage cell line.

The immunostimulatory properties of synthetic CpG oligodeoxynucleotides (ODNs) have been studied in various mammalian models including humans and mice. However, little was known about effects of CpG ODNs on immune responses of chickens, a common avian species with important economical value in the poultry industry. In the present study, two CpG ODNs, 2006 and 1826, which show immunomodulating properties for humans and mice were tested using a chicken macrophage cell line (HD11). ODN 2006, which has been reported to be an optimal stimulatory sequence for humans, showed strong immunomodulatory effects on HD11 cells, whereas ODN 1826, a CpG sequence with optimal immunostimulatory effects on mice, had weak influences on HD11 cells. ODN 2006 also induced strong IL-6 and nitric oxide secretion by HD11 cells in both dose- and time-dependent manners. Intracellular killing of Salmonella enteritidis (SE) was also increased in ODN 2006-activated HD11 cells. Furthermore, HD11 cells had reduced proliferation and underwent apoptosis, which is contradictory to the effects of ODN 2006 on human and murine cells. N(G)-monomethyl L-arginine (L-NMMA), an iNOS inhibitor, inhibited apoptosis of HD11 cells induced by ODN 2006, suggesting that this effect was likely mediated through an iNOS-dependent pathway. These results indicate that the differences in the responses of chicken HD11 macrophage cells to CpG ODNs compared to those of mammalian macrophages are species-related, and the potential of CpG ODNs as immunomodulators in poultry needs to be further explored.

Virulence testing of Listeria monocytogenes.

A major problem in understanding foodborne listeriosis from both the basic science and regulatory perspectives revolves around the role played by virulence factors of Listeria monocytogenes and how these interact with host susceptibility to result in the observed incidence of disease. From a mechanistic perspective, this problem has been well investigated, and many virulence components of L. monocytogenes have been discovered. Deletion of these genes results in large reductions in virulence functions in vitro and in vivo. The clonal bacteria and genetically identical hosts necessary to solve the riddles associated with virulence mechanisms are not likely to reflect the natural diversity found among wild populations of L. monocytogenes, including those associated with food. These factors contribute to a major dilemma in risk assessment and risk management of foodborne listeriosis: Although low-level L. monocytogenes contamination of certain foods is relatively common, suggesting widespread exposure, illness is overwhelmingly associated with only a relatively small subpopulation (3 of the 13 L. monocytogenes serotypes) and occurs in only a small proportion of susceptible individuals. Virulence testing based on DNA probes for virulence genes is confounded by the widespread distribution of these genes in food isolates. In terms of the distribution of virulence factors among food isolates of L. monocytogenes, only listeriolysin is well characterized, because beta-hemolysis is often used to confirm the presence of L. monocytogenes in foods. The presence of other virulence genes such as those involved in host cell invasion and cell-to-cell spread (inlA and actA) among food isolates has not been extensively investigated. How the presence of these components translates into functional virulence as measured in vivo and in vitro is also unknown. Animal studies and cell culture systems show a range of virulence among food isolates of L. monocytogenes. However, clinical isolates included in such studies are not consistently more virulent than food isolates with no known human disease association. Where multiple serotypes or ribotypes are compared, it has been difficult to demonstrate a consistent pattern of increased virulence associated with any subtype(s) in animal or in vitro studies. Development of model systems that adequately reflect the complexity of the host-pathogen relationship remains a challenge.

Potential allergenicity of novel proteins in murine models.

Bioengineered crops represent an important advancement for farmers who want to avoid losses caused by insect infestations or adverse environmental conditions. However, the use of modern biotechnology has raised questions regarding the safety of bioengineered foods because of the potential allergenicity of proteins expressed by the newly introduced genes. Standard approaches for safety assessment of these foods are still evolving. Animal models have been suggested as a tool that could help evaluate the potential allergenicity of such compounds. Several investigators are developing animal models to evaluate novel proteins, but none of these have yet been validated. This article reviews the published murine models, rat and mouse in particular, and the different methods used to evaluate parameters related to allergy. It also addresses the factors involved in the development of a model. Finally, it raises some questions that should be considered by the international community so that financial and intellectual efforts can be addressed in a unified manner.

Evaluation of extraction buffers using the current approach of detecting multiple allergenic and nonallergenic proteins in food.

The detection of food allergens has been a challenge because of the increasing need to ensure the absence of undeclared allergens in foods. The current trend in the detection of some food allergens, like peanuts, is based on the detection of multiple allergenic and nonallergenic proteins, and this is the approach that kit manufacturers have adopted. Because commercial kits differ in their ability to detect allergens, regulatory agencies, the food industry, and kit manufacturers are working together to standardize the detection methods. Three kits for the detection of peanuts have been evaluated for performance by the AOAC Research Institute. For this evaluation, a peanut butter suspension was used as a reference material. Several kit components contribute to between-kit analytical variation, even when the same sample is used. One component of commercial kits, which may be contributing to this variability, is the sample extraction buffer. In this study, differences in extractability of 3 allergenic foods were evaluated by using 4 different extraction buffers. The conclusion is that optimum allergen extractability was buffer-dependent, and no single buffer is appropriate for use as a universal extraction solution for all allergenic foods. Therefore, a thorough evaluation of sample preparation buffers needs to be performed for every individual allergenic food. In light of the results obtained, the current approach used for detection of peanut allergens based on the detection of multiple allergenic and nonallergenic proteins is being analyzed.

Effect of long chain fatty acids on Salmonella killing, superoxide and nitric oxide production by chicken macrophages.

The objective of this study was to investigate the effect of uptake of different commonly consumed long chain fatty acids on superoxide (O(2)(-)), nitric oxide (NO) production, and ability to kill Salmonella enterica serotype typhimurium (S. typhimurium) by chicken macrophages (HD11 cells). All the fatty acids were taken up by HD11 cells with stearic acid uptake higher than polyunsaturated fatty acids. Uptake of green fluorescent protein-labeled bacteria and the viability of HD11 cells (measured by flow cytometry) was not affected by any of the fatty acids tested. Bacterial clearance (measured by the plating of sorted viable infected cells) was significantly higher with n-3 fatty acids alpha-linolenic acid (ALA) and docosahexanoic acid (DHA). However, stearic acid (SA) and the n-6 fatty acid, arachidonic acid (ARA) did not influence S. typhimurium killing by HD11 cells. The improved S. typhimurium clearance by ALA and DHA was not associated with increased NO or O(2)(-) production by HD11 cells. These results suggest a role for n-3 polyunsaturated fatty acids in Salmonella clearance by chicken macrophages however in vivo studies are essential to confirm their efficacy in controlling Salmonella infection in chickens and contamination in shell eggs.

Impact of dietary components on chicken immune system and Salmonella infection.

Salmonella enterica serovars are facultative intracellular pathogens that may cause serious illness in poultry and humans. Human infection by two common serovars, Salmonella enteritidis (SE) and Salmonella typhimurium (ST) usually occurs via food-borne transmission. Consumption of raw or undercooked contaminated eggs usually causes SE infection, while ST is transmitted by contaminated chicken meat. There are several reports on dietary interventions, including fatty acid modifications, probiotic or prebiotic treatment on the immune system and/or Salmonella clearance in chickens. The aim of this review is to compile the information on the role of major dietary components on chicken immune system and Salmonella clearance. This may help design better poultry nutrition to lower Salmonella infection in chickens and, therefore, reduce human salmonellosis.

Live attenuated Listeria monocytogenes expressing HIV Gag: immunogenicity in rhesus monkeys.

Induction of strong cellular immunity will be important for AIDS vaccine candidates. Natural infection with wild-type Listeria monocytogenes (Lm), an orally transmitted organism, is known to generate strong cellular immunity, thus raising the possibility that live attenuated Lm could serve as a vaccine vector. We sought to examine the potential of live attenuated Lm to induce cellular immune responses to HIV Gag. Rhesus macaques were immunized with Lmdd-gag that expresses HIV gag and lacks two genes in the D-alanine (D-ala) synthesis pathway. Without this key component of the bacterial cell wall, vaccine vector replication critically depends on exogenous D-ala. Lmdd-gag was given to animals either solely orally or by oral priming followed by intramuscular (i.m.) boosting; D-ala was co-administered with all vaccinations. Lmdd-gag and D-ala were well tolerated. Oral priming/oral boosting induced Gag-specific cellular immune responses, whereas oral priming/i.m. boosting induced systemic as well as mucosal anti-Gag antibodies. These results suggest that the route of vaccination may bias anti-Gag immune responses either towards T-helper type 1 (Th1) or Th2 responses; overall, our data show that live attenuated, recombinant Lmdd-gag is safe and immunogenic in primates.

Ca2+ release from host intracellular stores and related signal transduction during Campylobacter jejuni 81-176 internalization into human intestinal cells.

Campylobacter jejuni is the leading bacterial cause of human diarrhoeal disease in many parts of the world, including the USA. The ability of C. jejuni to invade the host intestinal epithelium is an important determinant of virulence. A common theme among pathogenic invasive micro-organisms is their ability to usurp the eukaryotic cell-signalling systems both to allow for invasion and to trigger disease pathogenesis. Ca(2+) is very important in a great variety of eukaryotic cell-signalling processes (e.g. calmodulin-activated enzymes, nuclear transcriptional upregulation, and cytoskeletal rearrangements). This study analyses the effects of Ca(2+) availability on invasion of human INT407 intestinal epithelial cells by C. jejuni strain 81-176. The ability of C. jejuni to invade INT407 cells was not blocked by chelation of any remaining extracellular Ca(2+) from host cells incubated in Ca(2+)-free, serum-free media. In contrast, C. jejuni invasion was markedly reduced either by chelating host intracellular Ca(2+) with 1,2-bis-(2-)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA, AM) or by blocking the release of Ca(2+) from intracellular stores with dantrolene or U73122. Moreover, Bay K8644, a plasma-membrane Ca(2+)-channel agonist, was observed to stimulate C. jejuni invasion, presumably by increasing host intracellular free Ca(2+) levels. Measurement of host-cell cytosolic Ca(2+) via spectrofluorimetry and fluorescence microscopy revealed an increase in Ca(2+) from 10 min post-infection. Monolayer pretreatment with either a calmodulin antagonist or a specific inhibitor of protein kinase C was found to cause a marked reduction in C. jejuni invasion, suggesting roles for these Ca(2+)-activated modulators in signal-transduction events involved in C. jejuni invasion. These results demonstrate that C. jejuni induces the mobilization of Ca(2+) from host intracellular stores, which is an essential step in the invasion of intestinal cells by this pathogen.

Behavior and Viability of Third-Stage Larvae of Terranova sp. (Type HA) and Anisakis simplex (Type I) Under Coolant Conditions.

This study reports effects of storage at cold temperatures on behavior and survival of third-stage larvae of Terranova sp. (type HA) and Anisakis simplex (type I) in marine fishes. Snappers, caught near the Hawaiian Islands, were examined to determine whether type HA and type I larvae could migrate from the viscera of ungutted fishes into edible musculature when maintained at 12, 8, and 0°C. Our data are suggestive that both type HA and type I larvae possess the ability to migrate. Temperatures of 12, 8, and 0°C had no noticable adverse affect on viability of both larval types within fish tissues; however, both larval types were extremely sensitive to temperatures below freezing. Death of both larval types encysted within Hawaiian snappers occurred by day 4 at -5°C and within 24 h at -10, -15, and -20°C. Other type I larvae, collected from fishes ( Sebastes spp.) imported to Hawaii from the western Pacific, survived for slightly longer periods at -5, -10, -15, and -20°C when compared with type I larvae from Hawaiian fishes. Subjecting Hawaiian snappers to at least -20°C for 1 d and imported rockfishes to at least -20°C for 5 d is recommended to inactivate the living anisakines before ingesting any raw fish products.

Development and use of an ELISA test to detect IgE antibody to Cry9c following possible exposure to bioengineered corn.

BACKGROUND: Starlink(TM), a variety of corn genetically engineered to contain the insecticidal protein Cry9c, had not been approved for human consumption because it possessed some characteristics associated with allergenic proteins. However, in the fall of 2000 CRY9C DNA was detected in several corn-containing products, suggesting that Starlink corn had entered the human food supply. Subsequently, consumers, following consumption of corn products, reported a number of adverse health events, possibly consistent with allergic reaction. METHODS: To investigate the possibility of allergic reactions due to Cry9c in these consumers an ELISA test was developed for the purpose of detecting IgE antibodies to Cry9c and blood samples were taken from a total of 18 people who self-reported allergic reactions. Sera collected prior to the 1996 development of Starlink were used as negative controls. RESULTS: None of the adverse event sera were found to be reactive with recombinant Cry9c antigen, based on comparison with normal controls. Although a known human positive control serum containing IgE specific for Cry9c was not available, other controls were incorporated into the ELISA protocol, including the use of sera from subjects allergic to other allergens and their homologous antigens (cat, grass, peanut) to validate the IgE detection reagents. CONCLUSIONS: While the results do not support the likely occurrence of allergic reactions to Cry9c, such reactions cannot be ruled out, nor can the possibility that sera might react with unique glycosylated epitopes of Cry9c that may be expressed in the corn plant/seed.

Immune responses against Salmonella enterica serovar enteritidis infection in virally immunosuppressed chickens.

To understand the role of immune mechanisms in protecting chickens from Salmonella infections, we examined the immune responses of Salmonella enterica serovar Enteritidis-infected chickens and the effect of chicken anemia virus (CAV), a T-cell-targeted virus, on S. enterica serovar Enteritidis-induced immune responses. One-day-old chicks were orally inoculated with S. enterica serovar Enteritidis with or without intramuscular injection of CAV. The bacterial infection, pathology, and immune responses of chickens were evaluated at 14, 28, and 56 days postinoculation. The infection increased the levels of S. enterica serovar Enteritidis-specific mucosal immunoglobulin A (IgA), the number of gut-associated T cells, and the titer of serum IgG specific for S. enterica serovar Enteritidis surface antigens. CAV infection depressed these immune responses, especially the mucosal immune responses, but did not increase the number of S. enterica serovar Enteritidis-infected cells in the intestine. The severity of pathological lesions appeared to be reciprocal to the level of immune responses, but the S. enterica serovar Enteritidis infection persisted. These results suggest that oral infection of S. enterica serovar Enteritidis in chickens induces both mucosal and systemic immune responses, which have a limited effect on the S. enterica serovar Enteritidis infection under conditions designed to mimic the field situation.

Nonhuman primate model for Listeria monocytogenes-induced stillbirths.

Listeria monocytogenes, isolated from outbreaks in either human or nonhuman primate populations, was administered orally at doses ranging from 10(6) to 10(10) CFU. Four of 10 treated animals delivered stillborn infants. L. monocytogenes was isolated from fetal tissue, and the pathology was consistent with L. monocytogenes infection as the cause of pregnancy loss. For all pregnancies resulting in stillbirths, L. monocytogenes was isolated from maternal feces, indicating that L. monocytogenes had survived and had probably colonized the gastrointestinal tract. Antibodies and antigen-specific lymphocyte proliferation against Listeria increased in animals that had stillbirths.