Oral immunization against poliomyelitis

In the ten years since attenuated poliovirus was first administered to man for immunization against poliomyelitis, a vast body of experience has grown up and an attempt can now be made to assess the potentialities of oral poliovirus vaccine. The author of this paper seeks to answer two main questions: how safe are the strains now under study, and is oral vaccine effective in practice under the different conditions in which it may be used?From the information gained in large-scale trials involving a total of nearly 70 million people, he concludes that where poliomyelitis is predominantly a disease of infancy and early childhood the vaccines now available are safe both for the individual and for the community. The Sabin strains appear also to be safe for use in areas where the disease affects older children and adults; there is still not enough information to permit final conclusions to be drawn as to the safety of the Koprowski and Lederle strains in such areas.The evaluation of data on the efficacy of rural vaccines is beset with many problems, but the evidence of serological conversion rates suggests that, with one exception, there are no striking differences in efficacy between any of the strains when used in monovalent vaccines. However, 100% conversion cannot be expected in all circumstances from any oral vaccine given once only; repeated administration is clearly necessary, although it is still too early to say what the best vaccine schedule may be in any particular set of circumstances.While the problems yet to be solved are legion, great progress has nevertheless been made in the past decade, and the author looks forward to a steady extension of the use of live poliovirus vaccine and even to the eventual possibility of the eradication of poliomyelitis.     

Status of quality improvement activities to improve immunization practices and delivery: findings from the immunization quality improvement symposium, October 2003.

The Centers for Disease Control and Prevention convened a symposium on 22-23 October 2003 to bring together investigators and stakeholders working to apply the quality improvement (QI) approaches to immunization delivery in individual medical practices. The goal was to identify effective program components and further development of model programs. A call for projects was widely disseminated; of 61 submissions received, eight projects were selected. Three of the eight programs used the "train the trainer" approach, three used site-specific training, one used a "practice collaborative" approach, and one employed the use of tracking and outreach workers to effect change. At the symposium, invited experts reviewed each program. Common program features that appeared effective included involvement of a variety of staff within the office environment, collection and review of site-specific performance measurements to identify gaps in delivery, periodic monitoring of performance measurement to revise interventions and maintain the improvements, and provision of formal continuing education credits. While research is needed on ways to promote and integrate QI into practices, it is likely that a variety of QI strategies will be shown to be effective, depending on the clinical settings. The field will benefit from standardized outcome measures, cost analysis, and evaluation, so comparisons can be made among different programs.     

Oral immunization of rhesus macaques with adenoviral HIV vaccines using enteric-coated capsules

Targeted delivery of vaccine candidates to the gastrointestinal (GI) tract holds potential for mucosal immunization, particularly against mucosal pathogens like the human immunodeficiency virus (HIV). Among the different strategies for achieving targeted release in the GI tract, namely the small intestine, pH sensitive enteric coating polymers have been shown to protect solid oral dosage forms from the harsh digestive environment of the stomach and dissolve relatively rapidly in the small intestine by taking advantage of the luminal pH gradient. We developed an enteric polymethacrylate formulation for coating hydroxy-propyl-methyl-cellulose (HPMC) capsules containing lyophilized Adenoviral type 5 (Ad5) vectors expressing HIV-1 gag and a string of six highly-conserved HIV-1 envelope peptides representing broadly cross-reactive CD4⁺ and CD8⁺ T cell epitopes. Oral immunization of rhesus macaques with these capsules primed antigen-specific mucosal and systemic immune responses and subsequent intranasal delivery of the envelope peptide cocktail using a mutant cholera toxin adjuvant boosted cellular immune responses including, antigen-specific intracellular IFN-γ-producing CD4⁺ and CD8⁺ effector memory T cells in the intestine. These results suggest that the combination of oral adenoviral vector priming followed by intranasal protein/peptide boosting may be an effective mucosal HIV vaccination strategy for targeting viral antigens to the GI tract and priming systemic and mucosal immunity.     

Oral immunization of raccoons and skunks with a canine adenovirus recombinant rabies vaccine

Oral vaccination is an important part of wildlife rabies control programs. Currently, the vaccinia-rabies glycoprotein recombinant virus is the only oral rabies vaccine licensed in the United States, and it is not effective in skunks. In the current study, captive raccoons and skunks were used to evaluate a vaccine developed by incorporating the rabies virus glycoprotein gene into a canine adenovirus serotype 2 vector (CAV2-RVG). Seven of 7 raccoons orally vaccinated with CAV2-RVG developed virus neutralizing antibodies and survived lethal challenge. Five of 5 and 6 of 6 skunks in 2 experimental groups receiving 10-fold different dilutions of CAV2-RVG developed neutralizing antibodies and survived challenge. The results of this preliminary study suggest that CAV2-RVG stimulates protective immunity against rabies in raccoons and skunks.     

Oral immunization of Carassius auratus with modified recombinant A-layer proteins entrapped in alginate beads

This study was focused on the utilization of a recombinant expression system to produce a unique modified subunit vaccine possessing a self-contained delivery system which could potentially improve the uptake and delivery of vaccine products as well their immunogenic potential. For this purpose the A-layer protein (At-R) associated with the fish pathogen atypical Aeromonas salmonicida was cloned and modified by the genetic fusion of the protein transduction domain (MTS) derived from Kaposi fibroblast growth factor (At-MTS). The potential for these proteins to be employed as antigens for oral immunization of goldfish was examined by encapsulation of At-R, At-MTS and the control, BSA, into biodegradable alginate gel macrospheres which were fed to goldfish in place of standard pellet fish feed. The bead physical properties were modified only in the presence of At-R and the temporal release of proteins was significantly less when At-MTS was employed. Western blot analysis of serum samples collected from fish following intubation with the recombinant proteins determined that the rate of protein uptake from the digestive tract into the blood system improved considerably when MTS was fused to At-R. Experimental fish were fed one of three protein-alginate formulae on a schedule of 3 days/week or 5 days/month for a period of 2 months. After 1 month, animals fed on the 5-day protocol demonstrated increased serum antibody titers while following an additional month of feeding this level decreased and titers were found to be higher in fish maintained on the 3-day regime. Fish fed At-MTS maintained the highest titer at the end of 2-month period. To determine whether the diminished antibody titers were a result of oral tolerance fish were injected intraperitoneally with the At-R antigen. Only experimental groups which had been fed At-R or At-MTS demonstrated increased antibody titers which paralleled a typical secondary humoral response. In spite of the presence of an increased titer to A-protein, vaccinated fish did not demonstrate resistance to infection with atypical A. salmonicida.     

Oral immunization with recombinant Helicobacter pylori urease confers long-lasting immunity against Helicobacter felis infection

Recombinant Helicobacter pylori urease (rUre) has been shown to confer protection against challenge with Helicobacter felis in mice. The purpose of the present study was to examine duration of the immune response and long-term protective efficacy of immunization with rUre. Swiss Webster mice were orally immunized four times at weekly intervals with 100 microg rUre plus 5 microg heat-labile enterotoxin of Escherichia coli (LT) adjuvant, or with LT only. At 4, 10, 20 or 40 weeks post immunization, 25 rUre-immunized mice and control mice were challenged with H. felis and sacrificed at 2 or 10 weeks post-challenge. H. felis infection was assessed by gastric urease assay and by histology. Anti-H. pylori urease specific antibody levels were measured in serum and saliva both pre- and post-challenge. Over the 40 week time period, the infection rates in rUre-immunized mice were significantly lower than those in controls (p < 0.05) as assessed by gastric urease activity. Protection ranged from 79 100% at 2 weeks post-challenge and 63-78% at 10 weeks post-challenge. Gastric bacterial density in rUre-immunized mice was significantly lower than that of controls (p < 0.03) as determined by histologic assessment. Anti-urease antibody levels remained elevated in the serum and mucosal compartments at 39 weeks following immunization. This study shows that immunization with rUre plus LT results in long-lasting protective immunity against challenge with H. felis.     

Oral immunization of mice with temperature-sensitive Pseudomonas aeruginosa enhances pulmonary clearance of the wild-type

DBA/2J mice were immunized daily for 3 days per os with 10(8)-10(9) colony forming units (c.f.u.) of two different temperature-sensitive (TS) mutants of Pseudomonas aeruginosa. At varying times after the final immunization the animals were exposed to aerosols of the parental immunotype 1, and the ability of the immunized and control mice to clear their lungs of the wild-type (WT) challenge was measured 4 h later. The number of c.f.u. remaining in the lungs of mice immunized with one mutant, D/1/8, was significantly less (p less than 0.01) than the number remaining in the lungs of control mice and mice immunized with a second TS mutant, E/9/9.     

Oral immunization with a novel lipid-based adjuvant protects against genital Chlamydia infection

Oral immunization is attractive as a delivery route because it is needle-free and useful for rapid mass vaccination programs to target pandemics or bioterrorism. This potential has not been realized for human vaccination, due to the requirement of large antigen doses and toxic (to humans) adjuvants to overcome the induction of oral tolerance and potential degradation of antigens in the stomach. To date, only oral vaccines based on live attenuated organisms have been approved for human use. In this study we describe the use of a lipid-based delivery system/adjuvant, Lipid C, for oral immunization to protect mice against genital tract chlamydial infection. Lipid C is formulated from food-grade purified and fractionated triglycerides. Bacterial shedding following vaginal challenge with Chlamydia muridarum was reduced by 50% in female mice orally immunized with the chlamydial major outer membrane protein (MOMP) formulated in Lipid C, protection equivalent to that seen in animals immunized with MOMP admixed with both cholera toxin (CT) and CpG oligodeoxynucleotides (CpG-ODN). Protection was further enhanced when MOMP, CT and CpG were all combined in the Lipid C matrix. Protection correlated with production of gamma interferon (IFN) by splenic T cells, a serum MOMP-specific IgG response and low but detectable levels of MOMP-specific IgA in vaginal lavage.     

Oral immunization with size-purified microsphere beads as a vehicle selectively induces systemic tolerance and sensitization

Oral administration of antigens has long been recognized as a method to prevent or delay the onset of diseases associated with untoward immune responses to self and non-self antigens. Although oral administration of antigens offers a convenient way to induce systemic tolerance, its therapeutic potential has been seriously limited by the fact that it requires repeated feeding of a large amount of antigens and that it may deteriorate ongoing autoimmune diseases when autoantigens are employed. We have previously shown that orally administered poly-D,L-lactic acid (PDLLA) microspheres containing an antigen were selectively distributed to Peyer's patches (PP) and systemic lymphoid tissues according to their diameter and then released the antigen over a long period of time. We now report that a single dose of intragastric immunization with a PDLLA microsphere 7-10 micrometer in diameter and containing 2 mg of OVA was as effective as 100 mg of water soluble OVA to suppress OVA-specific IgG and DTH response. This was associated with a large increase of Interferon-gamma production by PPT cells stimulated with an antigen and a small increase in secretory IgA specific to OVA. In contrast, administration of an antigen encapsulated in microspheres 3-4 microm in diameter led to an enhanced OVA-specific IgG response and no significant increase in OVA-specific secretory IgA. Thus, by utilizing microspheres of an appropriate diameter as a vaccination vehicle, we were able to selectively induce both systemic tolerance and sensitization by oral ingestion of single low dose of an antigen.     

Oral immunization of mice with a multivalent therapeutic subunit vaccine protects against Helicobacter pylori infection

Helicobacter pylori is a human class I carcinogen and no effective prophylactic or therapeutic H. pylori vaccine has yet been marketed. H. pylori can escape the host immune response, but the precise immune protection mechanisms in humans remain unknown. In this study, we developed a multivalent, subunit H. pylori vaccine candidate by formulating three commonly used H. pylori antigens, neutrophil-activating protein (NAP), urease subunit A (UreA) and subunit B (UreB) with the mucosal adjuvant, a double-mutant heat-labile toxin (dmLT) from Escherichia coli, and evaluated its immunogenicity and therapeutic efficacy in a mouse model of H. pylori infection. We found that oral immunization of H. pylori-infected mice significantly reduced gastric bacterial colonization at both 2 and 8 weeks after immunization. The reduction in bacterial burdens was accompanied with significantly increased serum antigen-specific IgG responses and mucosal IgA responses. Moreover, oral immunization also induced Th1/Th17 immune responses, which may play a synergistic role with the specific antibodies in the elimination of H. pylori. Thus, our vaccine candidate appears able to overcome the immune evasion mechanism of H. pylori, restore the suppression of Th2 immune responses with the induction of a strong humoral immune response. These results lay the foundation for the development of an optimized oral therapeutic H. pylori vaccine with increased immunogenicity of UreA and UreB, as well as providing long-term immunity.     

Oral immunization of mice using transgenic tomato fruit expressing VP1 protein from enterovirus 71

Enterovirus 71 (EV71) causes seasonal epidemics of hand-foot-and-mouth disease associated with fatal neurological complications in young children, and several major outbreaks have occurred recently. This study developed an effective antiviral agent by transforming the gene for VP1 protein, a previously defined epitope and also a coat protein of EV71, into tomato plant. VP1 protein was first fused with sorting signals to enable it to be retained in the endoplasmic reticulum of tomato plant, and its expression level increased to 27 microg/g of fresh tomato fruit. Transgenic tomato fruit expressing VP1 protein was then used as an oral vaccine, and the development of VP1-specific fecal IgA and serum IgG were observed in BALB/c mice. Additionally, serum from mice fed transgenic tomato could neutralize the infection of EV71 to rhabdomyosarcoma cells, indicating that tomato fruit expressing VP1 was successful in orally immunizing mice. Moreover, the proliferation of spleen cells from orally immunized mice was stimulated by VP1 protein, and provided further evidence of both humoral and cellular immunity. Results of this study not only demonstrate the feasibility of using transgenic tomato as an oral vaccine to generate protective immunity in mice against EV71, but also suggest the probability of enterovirus vaccine development.     

Oral immunization of mice with ricin toxoid vaccine encapsulated in polymeric microspheres against aerosol challenge

Mucosal (oral) immunization of mice with carrier-delivered ricin toxoid (RT) vaccine was accomplished by one long (7 weeks) or two short (4 weeks) immunization schedules. For the long and short immunization schedule two lots of vaccine were administered prepared with the same procedure but at different occasions. The long schedule consisted of a total of seven doses of 50 microg of vaccine in microencapsulated (lot #108) or aqueous form administered on days 1, 2, 3, 28, 29, 30 and 49. With the short schedule a total of seven or six doses of 25 microg (lot #111) were administered on days 1, 2, 3, 14, 15, 16 and 30, or on 1, 2, 14, 15, 30, 31 and 32, respectively. Mice immunized orally with the long schedule, 50 microg of RT vaccine incorporated into poly-DL-lactide-co-glycolyde (DL-PLG) microspheres (MS) produced serum IgG, IgG2a and IgA ELISA antibodies. All mice immunized with RT in DL-PLG MS (RT-MS) were protected against a lethal ricin aerosol challenge. In contrast, with the same schedule and with the same dose, the aqueous vaccine (RT) failed to stimulate IgG, IgG2a and IgA antibodies, and these mice were not protected against an aerosol ricin toxin challenge. With the shorter immunization scheme, seven doses of 25 microg RT-MS stimulated a significant, though reduced, protection with the microencapsulated, but not with the aqueous vaccine. When the first and second 3-day cycles of the short immunization schedule was reduced to two doses, and the 3-day cycle was administered at the end of the schedule, neither RT-MS nor RT stimulated protection against the challenge. These results indicated that successful oral immunization with RT-MS depended on both the dose and the schedule, consisting of three consecutive days of administration in two cycles, 4 weeks apart. Altering this schedule and the dose, resulted in a reduced protection or no protection at all. Furthermore, under the conditions of this study, the advantage of the microencapsulated RT vaccine over the aqueous vaccine for effective oral immunization was well demonstrated.     

Oral immunization of adult volunteers with microencapsulated enterotoxigenic Escherichia coli (ETEC) CS6 antigen

As a step in the development of an oral vaccine against ETEC, we evaluated the safety and immunogenicity of CS6, a polymeric protein commonly found on the surface of ETEC. Formulations included 1 and 5mg doses of CS6, either encapsulated in biodegradable polymer poly(D, L)-lactide-co-glycolide (PLG), or as free protein, administered orally in a solution of either normal saline or a rice-based buffer. Three doses of CS6 were given at 2-week intervals. Blood was collected immediately before and 7 days after each dose. All formulations were well tolerated. Four of five volunteers who received 1mg CS6 in PLG microspheres with buffer had significant IgA ASC responses (median=30 ASC per 10(6) PBMC) and significant serum IgG responses (median=3.5-fold increase). Oral administration of these prototype ETEC vaccine formulations are safe and can elicit immune responses. The ASC, serum IgA, and serum IgG responses to CS6 are similar in magnitude to the responses after challenge with wild-type ETEC [Coster et al., unpublished data]. Further studies are underway to determine whether these immune responses are sufficient for protection.     

Oral DNA immunization in the second trimester fetal lamb and secondary immune responses in the neonate

We previously demonstrated that oral DNA vaccination of the third trimester fetus may be an effective strategy to prevent vertical disease transmission despite an immature immune system and the possibility of developing tolerance. The present investigation examined oral DNA vaccine delivery and immunogenicity in the second trimester fetal lamb (50-100 days of gestation (dg)). Histological examination revealed nucleated cells in the superficial layers of the oral mucosa. Therefore, luciferase-encoding DNA plasmid was injected into the oral cavity of 65-70 dg fetuses to monitor plasmid expression. Luciferase activity was detected in the oral mucosa of all fetuses but the level of luciferase activity varied among individual fetuses. Luciferase activity was also detected within the tonsils and lymph nodes draining the oral cavity. Oral DNA immunization between 55 and 83 dg with a truncated glycoprotein D (tgD)-encoding plasmid induced germinal centres in the draining lymph nodes and detectable tgD-specific serum antibody titers and/or IFNgamma-secreting cell responses in 16 of 24 (67%) fetuses. The tgD-specific antibody and IFNgamma responses persisted until birth in some fetuses but the magnitude of antibody titers in second trimester fetuses was low relative to antibody titers induced following oral DNA immunization in the third trimester. Lambs immunized during the second trimester of gestation responded to neonatal DNA immunization and anamnestic responses were detected in some lambs immunized as early as 67-72 dg. These observations confirmed that oral DNA immunization of the early second trimester fetus induced antigen-specific immune responses with no evidence of tolerance induction.     

Studies on vaccination against bacillary dysentery: 4. Oral immunization with live monotypic and combined vaccines

Results of tests made in 1964 confirm the previous findings that live oral vaccine, prepared from streptomycin-dependent strains of shigellae, confers a strong, type-specific protection against acute bacillary dysentery. This vaccine did not reduce the carrier rate of shigellae. Observations on soldiers treated with a vaccine of Shigella flexneri serotypes 2a and 3 combined revealed no antagonizing effects from the type 3 component upon the protective effect of the 2a component contained in the same vaccine.