Prospective study of systemic and mucosal immune responses in dysenteric patients to specific Shigella invasion plasmid antigens and lipopolysaccharides.

Shigellosis is a major cause of infant morbidity and mortality in developing countries. To find immunological correlates of specific protection against shigellosis, we examined chronological samples of sera, stool extracts, duodenal aspirates, and saliva samples from 39 adults and 22 children with shigellosis from Peru for the presence of specific antibody to invasion plasmid antigens (Ipa) common to all virulent Shigella strains, by using both a whole-organism enzyme-linked immunosorbent assay (ELISA) and a Western blot (immunoblot) assay. Antibody responses to lipopolysaccharide (LPS) from Shigella serotypes both homologous and heterologous to the infecting strain were also determined by ELISA. ELISAs showed that the highest serum immunoglobulin G (IgG) antibody titers to Shigella whole organisms both with and without surface Ipa were found in adults and malnourished children, the two groups with the shortest and longest durations of disease, respectively. Mucosal IgA antibody titers to Shigella strains decreased over time to a much greater extent than serum IgG titers, and IgA to Ipa in mucosal secretions was found in adults and well-nourished children but not in malnourished children. The presence of mucosal antibody to Ipa may limit the spread and severity of the infection, as indicated by the prolonged illness observed in malnourished children who have no significant mucosal antibody to Shigella Ipa. Serum antibody titers to the Ipa antigens were high relative to anti-Shigella LPS antibody titers, especially in pediatric patients. In contrast to the anti-Ipa responses observed, no differences in antibody responses to LPS in children compared by nutritional status were found. High levels of serum and mucosal cross-reacting antibody to heterologous serotype LPS were found between Shigella flexneri serotypes 1a and 2a. Different patterns of immune response to Ipa proteins and LPS that may aid in the definition of Shigella antigens important in host protection were observed in adults, well-nourished children, and malnourished children.     

Quantitation of serotype-specific and cross-reacting group-specific antigens by coagglutination and immunodiffusion tests for differentiating Actinobacillus (Haemophilus) pleuropneumoniae strains belonging to cross-reacting serotypes 3, 6, and 8.

There are strong cross-reactions among strains of Actinobacillus pleuropneumoniae belonging to serotypes 3, 6, and 8. Various serological tests were used to differentiate these serotypes from each other. Tube agglutination, coagglutination, and indirect hemagglutination tests were not sufficiently sensitive to differentiate strains of serotypes 3, 6, and 8. However, higher antibody titers were obtained with a 2-mercaptoethanol agglutination test in homologous rabbit antisera. Absorption of immune sera with homologous and heterologous serotypes as well as quantitative estimation of antigenic activity in the unheated and heat-treated bacterial cell suspensions of reference strains with rabbit homologous and heterologous antisera revealed serotype-specific and cross-reacting group-specific antigens. Usually, serotype-specific antigens were major and dominant over group-specific antigens. The coagglutination test could be used quantitatively to measure the ratio of serotype-specific and group-specific antigens with rabbit hyperimmune sera against serotypes 3, 6, and 8. The highest antigen content for a particular serotype reflected serotype-specific antigen. For strains showing equal amounts of antigen for two or more serotypes in the coagglutination test, the immunodiffusion test with boiled cell-saline extract as the antigen and rabbit antisera against whole-cell suspensions of serotypes 3, 6, and 8 clearly revealed the serotype-specific antigen. It is suggested that coagglutination and immunodiffusion tests could be used successfully to determine the exact serotype of strains belonging to serotypes 3, 6, and 8.     

Influence of Promoter, Gene Copy Number, and Preexisting Immunity on Humoral and Cellular Responses to a Vectored Antigen Delivered by a Salmonella enterica Vaccine

Attenuated Salmonella strains are currently in production as vaccines for protection of animals against salmonellosis. Such commercial strains offer the potential to deliver heterologous antigen to protect animals against other diseases. One vaccine strain, attenuated Salmonella enterica serovar Typhimurium (STM-1), was tested for the ability to deliver ovalbumin and to induce immune responses in mice. Two vaccine trials were performed testing the influence of promoter choice, the location of the encoding DNA (plasmid or chromosome), and the effect of preexisting homologous or heterologous immunity. The results demonstrated that humoral and T-cell responses were induced from either of two promoters, from either the plasmid or the chromosome, and that preexposure to the empty homologous vector, STM-1, or the heterologous vector, S. enterica serovar Enteritidis, had no detrimental effect on subsequent antigen-specific responses. In the case of homologous preexposure, responses were generally greater, and this was correlated with an increased uptake of Salmonella by macrophages in vitro after opsonization with immune sera.     

Enhanced respiratory clearance of nontypeable Haemophilus influenzae following mucosal immunization with P6 in a rat model.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of infection of the respiratory tract in children and adults. The search for an effective vaccine against this pathogen has focused on components of the outer membrane, and peptidoglycan-associated lipoprotein P6 is among the proposed candidates. This study investigated the immunogenicity of P6 in a rat respiratory model. P6 was purified from two strains of NTHi, one capsule-deficient strain and an H. influenzae type b strain, and assessed for clearance of both homologous and heterologous bacterial strains following mucosal immunization. A protective immune response was determined by enhancement of pulmonary clearance of live bacteria and an increased rate of recruitment of phagocytic cells to the lungs. This was most effective when Peyer's patch immunization was accompanied by an intratracheal (IT) boost. However, the rate of bacterial clearance varied between strains, which suggests some differences in anti-P6 immunological defenses recognizing the expression of the highly conserved P6 lipoprotein on the bacterial surface in some strains. P6-specific antibodies in both serum and bronchoalveolar lavage fluid were cross-reactive and did not differ significantly in strain specificity, demonstrating that difference in clearance was unlikely due to differences in P6-specific antibody levels. Serum homologous and heterologous P6-antibody was bactericidal against NTHi even when enhanced clearance had not been observed. Peyer's patch immunization induced P6-specific CD4+ T-helper cell proliferation in lymphocytes isolated from the mesenteric lymph nodes. An IT boost increased the level of P6-specific antibodies in serum and bronchoalveolar lavage fluid, and P6-specific mesenteric node lymphocyte proliferation. Cells from rats immunized with P6 demonstrated proliferation following stimulation with P6 from nonhomologous strains; however, there was some variation in proliferative responses to P6 from different strains in lymphocytes isolated from animals immunized with killed bacteria. The increase in P6-specific antibodies and T-helper cell responses following an IT boost correlated with an increased rate of recruitment of phagocytic cells and enhanced bacterial clearance of both homologous and heterologous bacteria in the lungs. The data suggests that P6 has the potential to afford protection against pulmonary infection by NTHi following the induction of effective antigen-specific B- and T-cell responses in mucosal tissues.     

Antigenic competition in the immune response against protein mixtures: strain-specific non-immunogenicity of Escherichia coli antigens

Antigenic competition is argued to impair the immune response on the level of accessory cell-dependent antigen presentation to responsive T-cells (regulated by MHC encoded Ir gene products). A possible influence of these mechanisms on in vivo immunization with antigen mixtures was investigated by using cytoplasmic extracts of four different strains of Escherichia coli as antigen sources for immunizing rabbits. The immune response against these antigen mixtures was tested by crossed immune electrophoresis (CIE) and immunoblotting (IB) in a homologous system (a given antigen extract of one strain against the corresponding antisera) and in the heterologous system (antigen extract of one strain against the antisera of different other strains). Several proteins were non-immunogenic in the extract of one strain but elicited good antibody responses in other strains. One of the strain-specific non-immunogenic proteins was purified and revealed a normal immune response upon immunization. The data suggest that different antigenic competition effects are induced by different protein compositions of antigen mixtures. This strain-specific competition seems to determine the immunogenicity of certain molecules (and not only the immunogenic properties of the molecules themselves). Furthermore this method offers a practical approach to increase the antibody production against weak immunogens in antigen mixtures.     

Inhibition of secondary anti-hapten responses with the hapten conjugated to type 3 pneumococcal polysaccharide

An injection of 2, 4-dinitrophenyl (DNP)-lysine conjugated to type 3 pneumococcal polysaccharide (DNP-lysine-SIII) markedly reduced the secondary IgG anti-DNP antibody response of irradiated mice injected with primed cells and the homologous dinitrophenylatcd foreign protein used for priming. Serum antibody titers against the foreign protein, hemocyanin, were not affected and the injection of DNP-lysine-SIII could be delayed until the 3rd day, but not the 4th day, after injection of the primed cells and homologous antigen. In mice in which anti-DNP antibody production was reduced by DNP-lysine-SIII, antibody isoelectric spectral analysis of residual antibody suggested that clones of antibody-producing cells were eliminated entirely or escaped unaltered. Purified SIII is a persisting antigen which readily induces macroglobulin responses in mice and does not seem to stimulate T cells. The results suggest that antigenic determinants on such antigens gain access to B cells and suppress IgC antibody production.     

Assessment of Antigen-Specific and Cross-Reactive Antibody Responses to an MF59-Adjuvanted A/H5N1 Prepandemic Influenza Vaccine in Adult and Elderly Subjects

Preparedness against an A/H5N1 influenza pandemic requires well-tolerated, effective vaccines which provide both vaccine strain-specific and heterologous, cross-clade protection. This study was conducted to assess the immunogenicity and safety profile of an MF59-adjuvanted, prepandemic influenza vaccine containing A/turkey/Turkey/01/2005 (H5N1) strain viral antigen. A total of 343 participants, 194 adults (18 to 60 years) and 149 elderly individuals (≥61 years), received two doses of the investigational vaccine given 3 weeks apart. Homologous and heterologous antibody responses were analyzed by hemagglutination inhibition (HI), single radial hemolysis (SRH), and microneutralization (MN) assays 3 weeks after administration of the first vaccine dose and 3 weeks and 6 months after the second dose. Immunogenicity was assessed according to European licensure criteria for pandemic influenza vaccines. After two vaccine doses, all three European licensure criteria were met for adult and elderly subjects against the homologous vaccine strain, A/turkey/Turkey/1/2005, when analyzed by HI and SRH assays. Cross-reactive antibody responses were observed by HI and SRH analyses against the heterologous H5N1 strains, A/Indonesia/5/2005 and A/Vietnam/1194/2004, in adult and elderly subjects. Solicited local and systemic reactions were mostly mild to moderate in severity and occurred less frequently in the elderly than in adult vaccinees. In both adult and elderly subjects, MF59-adjuvanted vaccine containing 7.5 μg of A/Turkey strain influenza virus antigen was highly immunogenic, well tolerated, and able to elicit cross-clade, heterologous antibody responses against A/Indonesia and A/Vietnam strains 6 weeks after the first vaccination.     

Respiratory syncytial virus group-specific antibody response in nasopharyngeal secretions from infants and children after primary infection.

Respiratory syncytial virus (RSV) group-specific immunoglobulin A (IgA) and IgG enzyme-linked immunosorbent assay antibody and neutralizing antibody responses were determined for nasopharyngeal secretions (NPS) from 27 infants and children (6 to 18 months of age) undergoing primary infection with RSV group A or B strain. IgA and IgG antibody responses against RSV envelope glycoproteins (fusion [F] and large [G] glycoprotein) in NPS were also analyzed. Most subjects examined developed moderate levels of NPS IgA and IgG antibodies and neutralizing antibody activity to both group A and B strains in convalescent phase; however, the levels of antibodies to homologous strains were significantly higher than to the heterologous strains. Patients infected with group A developed antibodies in both F and G glycoproteins of A2 strains (group A). Patients infected with group B developed levels of antibody activity to F glycoprotein of A2 strain similar to those of patients infected with group A. However, these subjects developed little or no antibody response to G glycoprotein of A2 strain. These data suggest that the IgA and IgG antibody responses to G glycoprotein in the respiratory tract are group specific. It is suggested that lack of antibody response to the G glycoprotein of the heterologous group in the respiratory tract may determine the outcome of reinfection with other RSV strains.     

Local and systemic immune responses in humans againstHelicobacter pyloriantigens from homologous and heterologous strains

The capacity ofHelicobacter pylorito induce strain specific immune responses was studied in adult Swedish volunteers. Sera and gastric aspirates from 11H. pylori-infected subjects were tested for specific antibody levels against, respectively, lipopolysaccharides (LPS) and total membrane preparations (MPs) prepared from the study subjects» own strains, as well as with corresponding antigens from two referenceH. pyloristrains or heterologous strains collected from other subjects within the study. It was found that sera from five of the 11 subjects had significantly higher IgA antibody titres against LPS from the homologous strain than against LPS from either of the reference strains and in five cases sera reacted with higher IgG titres against the homologous LPS than with LPS preparations from the reference or heterologous patient strains. Analyses of specific titres against MPs revealed that six sera had higher IgA titres and four sera had higher IgG titres against MPs prepared from the subjects» own strains than against MPs from either of the two reference strains. Determination of specific antibodies in gastric aspirates revealed significantly higher IgA titres against LPS from the homologousH. pyloriisolate than against LPS from the two reference strains in five cases, and six aspirates reacted in higher IgA titre with the homologousH. pyloriMPs. Results from immunoblotting analyses of sera support induction of strain specific immune responses againstH. pyloriLPS. By means of specific monoclonal antibodies againstH. pyloriLPS, antigenic heterogeneity between the different LPS preparations tested was confirmed.     

Production of nontypeable Haemophilus influenzae HtrA by recombinant Bordetella pertussis with the use of filamentous hemagglutinin as a carrier

Bordetella pertussis, the etiologic agent of whooping cough, is a highly infectious human pathogen capable of inducing mucosal and systemic immune responses upon a single intranasal administration. In an attenuated, pertussis toxin (PTX)-deficient recombinant form, it may therefore constitute an efficient bacterial vector that is particularly well adapted for the delivery of heterologous antigens to the respiratory mucosa. Filamentous hemagglutinin (FHA) has been used as a carrier to present foreign antigens at the bacterial surface, thereby inducing local, systemic, and protective immune responses to these antigens in mice. Both full-length and truncated (Fha44) forms of FHA have been used for antigen presentation. To investigate the effect of the carrier (FHA or Fha44) on antibody responses to passenger antigens, we genetically fused the HtrA protein of nontypeable Haemophilus influenzae to either FHA form. The fha-htrA and Fha44 gene-htrA hybrids were expressed as single copies inserted into the chromosome of PTX-deficient B. pertussis. Both chimeras were secreted into the culture supernatants of the recombinant strains and were recognized by anti-FHA and anti-HtrA antibodies. Intranasal infection with the strain producing the FHA-HtrA hybrid led to significantly higher anti-HtrA and anti-FHA antibody titers than those obtained in mice infected with the Fha44-HtrA-producing strain. Interestingly, the B. pertussis strain producing the Fha44-HtrA chimera colonized the mouse lungs more efficiently than the parental, Fha44-producing strain and gave rise to higher anti-FHA antibody titers than those induced by the parental strain.     

Cross-protection and antigen expression by chicken embryo-grown Pasteurella multocida in chickens

The growth of Pasteurella multocida strain X-73 (serotype 1) and P-1059 (serotype 3) in vivo instead of in vitro resulted in the expression of additional antigens, as revealed by SDS-PAGE profile and western blotting of the outer membrane detergent-insoluble fraction (DIF-OM). Minor protein bands ranging from 58-138 kDa were expressed by both strains; major bands ranging from 35-64 and 35-43 kDa were expressed by X-73 and P-1059, respectively. Antigens at 35-39 kDa were dense and dominant. Treatment of DIF-OM from both strains with sodium periodate and proteinase K, but not with trypsin, reduced ELISA reactivity; this suggested that DIF-OM was composed of glycoprotein. Western blotting with heterologous antisera demonstrated antigenic cross-reactivity between strains X-73 and P-1059 grown in vivo. Inactivated vaccine prepared from X-73 grown in vivo protected chickens against challenge with X-73, P-1059 and strain P-1662 (serotype 4); a similar vaccine prepared from in-vitro culture, however, protected only against the homologous serotype. Moreover, antiserum against X-73 grown in vivo but not against the same strain grown in vitro gave considerable passive protection against challenge with the heterologous strains P-1059 and P-1662.     

Serological study of trichloroacetic acid extracts of Bacteroides fragilis.

Immunodiffusion techniques were used on trichloroacetic acid extracts from 10 strains of Bacteroides fragilis in detecting precipitating antibodies against this species in immune rabbit sera. Species and even strain specificities were observed in these precipitin reactions. Multiple antigens were detected in the extracts from some strains, whereas only one precipitin band per extract developed during agar-gel diffusion tests of others. The antigen extracts were found to be both heat stable and resistant to hydrolysis by alpha-chymotrypsin. Four serological patterns were demonstrated in homologous and heterologous reactions with the B. fragilis. antigen-antibody systems used. The results showed that some strains were serologically distinct from others, indicating that the strains tested are of more than one serotype.     

Host and bacterial factors affecting induction of immune responses to flagellin expressed by attenuated Salmonella vaccine strains

Previous observations demonstrated that the delivery of recombinant Salmonella enterica serovar Dublin strains to mice via mucosal routes did not efficiently activate systemic and secreted antibody responses to either type d flagellin or genetically fused heterologous B-cell epitopes, thus reducing the usefulness of the protein as a carrier of epitopes for vaccine purposes. In this work, we investigated murine systemic and mucosal flagellin immunogenicity after oral immunization with attenuated Salmonella strains. The reduced anti-type d flagellin antibody responses in mice immunized via mucosal routes with three doses of flagellated S. enterica serovar Dublin strains were not caused by oral tolerance and could not be restored by coadministration of a mucosal adjuvant. The induction of antibody responses to Salmonella flagellins was shown to differ according to the genetic background, but not the haplotype, of the mouse lineage. Moreover, BALB/c mice orally immunized with S. enterica serovar Typhimurium strains developed anti-type i flagellin sera and secreted antibody responses, which indicated that the serovar of the Salmonella vaccine strain also affected flagellin immunogenicity. Analyses of cytokine responses of BALB/c mice immunized with three oral doses of flagellated S. enterica serovar Dublin vaccine strains showed that, in spite of the lack of antibody responses, elevated type d flagellin-specific CD4-cell-activation-dependent gamma interferon (IFN-gamma) and interleukin-10 responses were elicited after the administration of the vaccine strains via either parenteral or mucosal routes. Similar cytokine production patterns were detected to a T-cell heterologous epitope, derived from the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC), in mice orally immunized with a Salmonella vaccine strain expressing hybrid flagella. These results indicate that the immunogenicities of Salmonella flagellins can differ significantly, depending on the murine host and on the bacterial vector used, and demonstrate that the induction of CD4-cell-activation-dependent IFN-gamma production represents a major immune response triggered by flagellin and in-frame fused heterologous T-cell epitopes after the oral administration of recombinant S. enterica serovar Dublin vaccine strains.     

Comparative Antigenicity and Immunogenicity of A/USSR/77 Influenza Vaccines in Normal and Primed Mice

In response to the threat of wide-spread epidemics of influenza in the United States due to the A/USSR/77 strain of virus, vaccines containing A/USSR/77 virus were prepared by four manufacturers. The vaccines were standardized by immunological measurements of viral hemagglutinin and were tested for their ability to induce serum hemagglutination-inhibiting antibodies in mice and to protect the animals against challenge infection with A/USSR/77 virus. Whole-virus and subunit virus vaccines were found to be equally efficacious, in contrast to our previous findings with vaccines prepared from other influenza virus strains. The effect of priming animals by infection with representative viruses of earlier eras on their response to A/USSR/77 vaccines was also studied. Enhanced responses were noted to both subunit and whole-virus A/USSR/77 vaccines in animals primed with viruses prevalent before 1957; higher antibody titers were induced with the subunit vaccine. A high degree of heterologous protection to A/USSR/77 challenge infection was found in mice primed with virus strains having hemagglutinin antigens unlike those of A/USSR/77 virus. Comparison of the responses of mice with those of humans inoculated with the same vaccines showed a similarity in many instances, with higher responses in those individuals primed with H0N1 and H1N1 viruses than in younger vaccinees.     

Precipitating antigens associated with Marek's disease viruses and a herpesvirus of turkeys

Precipitating antigens associated with a number of Marek's disease virus strains and with a turkey herpesvirus have been analyzed. The 'A' antigen has been defined as the major soluble antigen in feather follicles of infected chickens, which is identical with the major antigen usually present in supernatants of chicken kidney cell cultures infected with strains of Marek's disease virus. 'BC' antigens are 2 or more antigens which are usually not noted in skin extracts but present in cultured cells infected with Marek's disease virus or turkey herpesvirus, in addition to the 'A' antigen. Some of the virus strains examined were positive and others negative for 'A' antigen, but all contained the 'BC' antigens. Results of agar-gel precipitin tests suggested a serological classification of the group of avian herpesviruses formed by Marek's disease viruses and turkey herpesvirus into 3 types. Pathogenic strains of Marek's disease virus and their attenuated A- variants, represented by the HPRS-16 strain (HPRS-16, JM, GA, VC, Oldenburg). Apathogenic Marek's disease virus, represented by the HPRS-24 strain. Turkey herpesvirus and its A- variants, represented by the FC126 strain. A serological subdivision corresponding to the different grades of pathogenicity of virus strains of the first type was not possible. Differences between antigens associated with the 3 types of virus were apparent from the antigen and antibody titres against homologous and heterologous reagents. Precipitin bands produced by homologous antigen and antibody were stronger than those produced by heterologous reagents. Differences between 'A' antigens of the 3 virus types were characterized by spur patterns of precipitin bands indicating a partial identity. At least 3 'BC' precipitin bands were noted; at least one was group-specific and one appeared to be type-specific.     

Attenuated Salmonella enterica serovar Typhi expressing urease effectively immunizes mice against Helicobacter pylori challenge as part of a heterologous mucosal priming-parenteral boosting vaccination regimen

Recombinant vaccine strains of Salmonella enterica serovar Typhi capable of expressing Helicobacter pylori urease were generated by transforming strains CVD908 and CVD908-htrA with a plasmid harboring the ureAB genes under the control of an in vivo-inducible promoter. The plasmid did not interfere with the ability of either strain to replicate and persist in human monocytic cells or with their transient colonization of mouse lungs. When administered to mice intranasally, both recombinant strains elicited antiurease immune responses skewed towards a Th1 phenotype. Vaccinated mice exhibited strong immunoglobulin G2a (IgG2a)-biased antiurease antibody responses as well as splenocyte populations capable of proliferation and gamma interferon (IFNgamma) secretion in response to urease stimulation. Boosting of mice with subcutaneous injection of urease plus alum enhanced immune responses and led them to a more balanced Th1/Th2 phenotype. Following parenteral boost, IgG1 and IgG2a antiurease antibody titers were raised significantly, and strong urease-specific splenocyte proliferative responses, accompanied by IFNgamma as well as interleukin-4 (IL-4), IL-5, and IL-10 secretion, were detected. Neither immunization with urease-expressing S. enterica serovar Typhi alone nor immunization with urease plus alum alone conferred protection against challenge with a mouse-adapted strain of H. pylori; however, a vaccination protocol combining both immunization regimens was protective. This is the first report of effective vaccination against H. pylori with a combined mucosal prime-parenteral boost regimen in which serovar Typhi vaccine strains are used as antigen carriers. The significance of these findings with regard to development of a human vaccine against H. pylori and modulation of immune responses by heterologous prime-boost immunization regimens is discussed.     

Host defenses in experimental scrub typhus: role of cellular immunity in heterologous protection.

The relative contributions of cellular and humoral immunity in scrub typhus infections were studied in inbred mice employing paired strains of Rickettsia tsutsugamushi differing in virulence. An infectious dose (100 MID50) of the less virulent Gilliam strain resulted in heterologous immune protection against an otherwise lethal challenge (1,000 MLD50) of the virulent Karp strain. Partial heterologous protection against lethal Karp challenge was observed in animals preimmunized with the Gilliam strain as early as 3 days prior to challenge, whereas complete protection against illness and death existed in animals immunized at least 7 days prior to challenge. In the heterologous protection provided by prior Gilliam infection, the role of humoral immunity was not of primary importance for the following reasons: (i) significant levels of complement-fixing antibody against R. tsutsugamushi were not detectable until long after animals were solidly immune; (ii) antibody eventually appearing after Gilliam immunization exhibited a consistently low complement-fixing titer against the immunizing homologous (Gilliam) strain and contained no detectable activity against the heterologous challenge (Karp) strain; and (iii) passive transfer of large quantities of serum from Gilliam immune mice, themselves immune to Karp challenge, failed to protect recipients against a similar challenge. However, protection was afforded by the passive transfer of serum containing antibody against Karp, suggesting a major role for antibody in protection against homologous infection. This heterologous challenge system was particularly useful because it minimized the role of humoral immunity, at least early in the course of infection, and allowed a definitive examination of the cellular response. Cell-mediated immunity played a major role in the heterologous protection observed after Gilliam immunization. This was evidenced by the significant protection against Karp challenge afforded by the passive transfer of spleen cells from animals immunized with Gilliam 7 to 63 days previously. Of the immune spleen cells, only those which were nonadherent, presumably lymphocytes, were capable of transferring passive heterologous protection. This protective effect of nonadherent cells could be ablated by depleting the cell population of thymus-derived or T cells with anti-theta serum and complement prior to transfer but not by use of anti-immunoglobulin serum and complement, which selectively removes bone marrow-derived or B cells. These results suggested that the cell in immune spleens capable of conferring heterologous protection was a T lymphocyte.     

Salmonella flagellin fused with a linear epitope of colonization factor antigen I (CFA/I) can prime antibody responses against homologous and heterologous fimbriae of enterotoxigenic Escherichia coli

In this work, a 15-amino-acid-long peptide derived from the enterotoxigenic Escherichia coli CFA/I fimbria (11VDPVIDLLQADGNAL25) was genetically fused to the Salmonella flagellin and used to prime and boost serum antibody responses (IgG) against homologous (CFA/I) and heterologous (CS1) colonization factors (CFs) in BALB/c mice. Antibodies raised against the hybrid flagellin (Fla II) cross-reacted with CFA/I, CS1, CS2, and PCFO166 but not with CS4. Parenteral administration of Fla II primed antibody responses against both CFA/I and CS1 but boosted IgG responses only against CFA/I. These findings confirm that linear epitopes derived from the CFA/I fimbria can prime antibody responses against homologous and heterologous CFs and indicate that Salmonella flagellin represents a potential carrier for the development of broad-range peptide-based anti-colonization ETEC vaccines.     

Genetic control of antibody responses induced against an antigen delivered by recombinant attenuated Salmonella typhimurium.

Recombinant derivatives of nonpathogenic bacteria such as attenuated Salmonella typhi have the potential to be used for delivery of heterologous antigens to the immune system. Genetic factors may modulate the immune responses to these live attenuated organisms and could therefore modify the immunogenicity of future human vaccines. In the present study, we compared the antibody responses of Ity or H-2 congenic strains of mice to a foreign antigen expressed by the murine attenuated aroA S. typhimurium strain. Our results demonstrate that the Ity gene may modulate the antibody responses to the foreign antigen but that the major genetic influence is exerted by H-2 genes, which control the capacity of mice to respond to the antigen expressed by recombinant attenuated Salmonella cells. This genetic control is related to differences in responsiveness of different strains of mice to low doses of antigen. Increasing the amount of foreign antigen expressed by recombinant Salmonella cells overcame the genetic restriction of these responses. These findings are potentially of great importance for the design of live vaccines for humans and show that care must be taken to optimize the amount of foreign antigen delivered to the immune system.     

Protection against experimental Helicobacter pylori infection after immunization with inactivated H. pylori whole-cell vaccines

The protective effect of therapeutic oral immunization with homologous and heterologous formalin-inactivated Helicobacter pylori cells given together with cholera toxin as an adjuvant was evaluated with C57BL/6 mice infected with H. pylori Sydney strain 1 (SS1). The bacteria used for immunization were strains that were either homologous or heterologous with regard to the O antigen (i.e., the Lewis antigen [Le antigen]) expressed by the lipopolysaccharide of the infecting H. pylori SS1 strain. We found that repeated oral immunization with inactivated H. pylori SS1 cells can significantly inhibit an existing infection (P < 0.001) and that the protection induced by such therapeutic immunization extends to protection against reinfection (P < 0.001). A similar level of protection was also achieved by immunization with another inactivated H. pylori strain having the same O antigen (Le antigen) as the infecting H. pylori SS1 strain. In contrast, immunization with inactivated strains expressing a heterologous O antigen, Le(x), provided less protection or no protection. Immunization with H. pylori lysate preparations, on the other hand, resulted in significant comparable protection whether the lysates were prepared from an Le(x) strain or an Le(y) strain. Postimmunization gastritis was seen in mice that were protected after vaccination but not in unimmunized or unprotected mice. In conclusion, therapeutic immunization with inactivated H. pylori whole-cell vaccines may provide strong protection both against experimental H. pylori infection and against later reinfection.