Compared with vaccine delivery by injection, oral vaccines offer the hope of more convenient immunization strategies and a more practical means of implementing universal vaccination programs throughout the world. Oral vaccines act by stimulating the immune system at effector sites (lymphoid tissue) located in the gut. Genetic engineering has been used with variable success to design living and non-living systems as a means to deliver antigens to these sites and to stimulate a desired immune response. More recently, plant biotechnology techniques have been used to create plants which contain a gene derived from a human pathogen; the resultant plant tissues will accumulate an antigenic protein encoded by the foreign DNA. In pre-clinical trials, we found that antigenic proteins produced in transgenic plants retained immunogenic properties when purified; if injected into mice the antigen caused production of protein-specific antibodies. Moreover, in some experiments, if the plant tissues were simply fed to mice, a mucosal immune response occurred. The present study was conducted as a proof of principle to determine if humans would also develop a serum and/or mucosal immune response to an antigen delivered in an uncooked foodstuff.

Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato.

Animal models of Salmonella infections: enteritis versus typhoid fever

M cells are specialized epithelial cells of the mucosa-associated lymphoid tissues. A characteristic of M cells is that they transport antigens from the lumen to cells of the immune system, thereby initiating an immune response or tolerance. Soluble macromolecules, small particles, and also entire microorganisms are transported by M cells. The interactions of these substances with the M cell surface, their transcytosis, and the role of associated lymphoid cells are reviewed in detail. The ultrastructure and several immuno- and lectin-histochemical properties of M cells vary according to species and location along the intestine. We present updated reports on these variations, on identification markers, and on the origin and differentiation of M cells. The immunological significance of M cells and their functional relationship to lymphocytes and antigenpresenting cells are critically reviewed. The current knowledge on M cells in mucosa-associated lymphoid tissues outside the gut is briefly outlined. Clinical implications for drug deliver, infection, and vaccine development are discussed.

M cells in Peyer's patches of the intestine.

In the middle 80s, recombinant hepatitis B virus cores (HBc) gave onset to icosahedral virus-like particles (VLPs) as a basic class of non-infectious carriers of foreign immunological epitopes. The recombinant HBc particles were used to display immunodominant epitopes of hepatitis B, C, and E virus, human rhinovirus, papillomavirus, hantavirus, and influenza virus, human and simian immunodeficiency virus, bovine and feline leukemia virus, foot-and-mouth disease virus, murine cytomegalovirus and poliovirus, and other virus proteins, as well as of some bacterial and protozoan protein epitopes. Practical applicability of the HBc particles as carriers was enabled by their ability to high level synthesis and correct self-assembly in heterologous expression systems. The interest in the HBc VLPs was reinforced by the resolution of their fine structure by electron cryomicroscopy and X-ray crystallography, which revealed an unusual alpha-helical organization of dimeric units of HBc shells, alternative packing into icosahedrons with T = 3 and T = 4 symmetry, and the existence of long protruding spikes. The tips of the latter seem to be the optimal targets for the display of foreign sequences up to 238 amino acid residues in length. Combination of numerous experimental data on epitope display with the precise structural information enables a knowledge-based design of diagnostic, and vaccine and gene therapy tools on the basis of the HBc particles.

HBV Core Particles as a Carrier for B Cell/T Cell Epitopes

The phoP/phoQ virulence regulatory genes of Salmonella typhi Ty2 were deleted, and the resultant strain (Ty800) was tested as a live attenuated typhoid fever vaccine in human volunteers. Groups of 2 or 3 subjects received single oral doses of 10 7 , 10 8 , 10 9 , or 10 10 cfu. Two volunteers who received the largest dose had self-limited side effects ; no bacteremias were detected. Ten of 11 subjects had evidence of intestinal immune responses to the vaccine as measured by increases in S. typhi lipopolysaccharide-specific IgA-secreting cells in peripheral blood samples. Humoral immune responses were measured and compared with those of control vaccinees who received 4 oral doses of S. typhi Ty21a. In the most sensitive assays, 9 of 11 volunteers and 5 of 8 Ty21a control vaccinees had evidence of IgG directed against S. typhi antigens. Ty800 is safe, and single oral doses are highly immunogenic in humans.

phoP/phoQ-Deleted Salmonella typhi (Ty800) Is a Safe and Immunogenic Single Dose Typhoid Fever Vaccine in Volunteers

Three attenuated Salmonella typhi strains have been constructed by introducing deletions in aroC and aroD or deletions in cya and crp into one of two wild-type parent strains, Ty2 or ISP1820. These mutant strains were designated CVD 906 (ISP1820 delta aroC delta aroD), CVD 908 (Ty2 delta aroC delta aroD), and chi 3927 (Ty2 delta cya delta crp). Two studies were conducted with 36 healthy adult inpatient volunteers to determine in a double-blind fashion the safety and immunogenicity of approximately 5 x 10(4) and 5 x 10(5) CFU of each of these three vaccine candidates given as a single dose. No statistically significant difference in the incidence of reactions among vaccinees was observed. Fever (oral temperature greater than or equal to 38.2 degrees C) occurred in 2 of 12 volunteers who received CVD 906, in 0 of 12 who received CVD 908, and in 1 of 12 who received chi 3927. Vaccine bacteremia without symptoms occurred in 1 of 12 vaccinees who received CVD 906, in 0 of 12 who received CVD 908, and in 2 of 12 who received chi 3927. Overall, 19 (53%) of 36 vaccinees developed immunoglobulin G antibody to S. typhi lipopolysaccharide after vaccination, with no statistically significant differences in the rate of seroconversion among volunteers in the three groups. We conclude that defined mutations in the aromatic biosynthetic pathway and in the cyclic AMP global regulatory system attenuate S. typhi. Mutant strains CVD 906, CVD 908, and chi 3927 are highly (and approximately equally) immunogenic but possibly differ in their propensity to induce fever. Further studies are needed to document the apparent relative safety of CVD 908 as a typhoid vaccine and as a vaccine carrier of foreign antigens.

https://iai.asm.org/content/iai/60/2/536.full.pdf

Comparison of the safety and immunogenicity of delta aroC delta aroD and delta cya delta crp Salmonella typhi strains in adult volunteers.

Attenuated Salmonella typhi organisms which express genes encoding protective antigens of other pathogens have been developed for use as experimental oral vaccines. A delta asd S. typhi strain attenuated by deletions in cya, crp, and cdt which contains hepatitis B core (HBc) and pre-S genes encoded on an Asd+ pBR-based plasmid vector was constructed. Healthy adult volunteers ingested a single dose of 5 x 10(5) to 5 x 10(8) CFU of strain chi4073 (delta cya delta crp delta cdt S. typhi Ty2), 6 x 10(7) or 1 x 10(9) CFU of strain chi4632(pYA3149), a further derivative of chi4073 deleted in asd and containing the Asd+ vector without the HBc-pre-S fusion, or 3 x 10(7) or 7 x 10(8) CFU of strain X4632(pYA3167), a derivative containing the vector with the HBc-pre-S fusion. Chi4073 was generally well tolerated by 22 volunteers. No volunteer had fever or positive blood cultures; 4 of 22 volunteers shed vaccine organisms in the stool in the first 48 h only. Two of 18 volunteers who received one of the plasmid-containing derivatives of chi4073 developed low-grade fevers on day 10 or 12 after ingestion. One of these volunteers had positive blood cultures on days 7 and 8. Seven of these 18 volunteers had vaccine organisms detected in their stools in the first 48 h only. Most volunteers developed S. typhi-specific serum responses and developed S. typhi-specific antibody-secreting cells. However, no volunteer developed serum antibody to hepatitis pre-S or pre-S-specific antibody-secreting cells. Although the parent strain chi4073 was well tolerated, induced immunoglobulin G seroconversion to S. typhi lipopolysaccharide in 80 to 100% of vaccinees and stimulated specific IgA-secreting lymphocytes in 80 to 100% of vaccinees given a single oral dose of 2 x 10(7) and 5 x 10(8) CFU, chi4073 derivatives containing the Asd+ vector with and without sequences encoding the HBc-pre-S fusion caused occasional febrile reactions at high doses and did not stimulate detectable immune responses to hepatitis B antigens.

https://iai.asm.org/content/iai/65/8/3381.full.pdf

Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system.

Erratum: Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system (Infection and Immunity 65:8 (3383))

S. M. Kelly

Papers

Comparison of the safety and immunogenicity of delta aroC delta aroD and delta cya delta crp Salmonella typhi strains in adult volunteers.

Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system.

Erratum: Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system (Infection and Immunity 65:8 (3383))