Oral immunization using live attenuated Salmonella spp. as carriers of foreign antigens.

A variety of techniques, including the use of live oral vaccines, have been used to deliver antigens to the gut-associated lymphoid tissues in an attempt to initiate production of specific secretory immunoglobulin A for protection against pathogens that colonize or cross mucosal surfaces to initiate infection. A number of attenuated Salmonella mutants are able to interact with the lymphoid tissues in the Peyer's patches but are not able to cause systemic disease. Some of these mutants are effective as live vaccines (i.e., able to protect against infection with the virulent Salmonella parent) and are candidates for use as carriers for virulence determinants of other mucosal pathogens. This has been shown to be an effective means of stimulating significant levels of specific mucosal secretory immunoglobulin A directed against the carrier strains and against a variety of heterologous antigens and has been shown to stimulate production of serum antibodies and cell-mediated responses as well. This review examines the history of this mechanism of vaccine delivery and summarizes the most recent applications of this evolving technology. This is a technique for vaccine delivery with significant potential for influencing the management of infectious diseases on a large scale. It can be used not only for vaccines against enteric bacterial pathogens but also for vaccines against a variety of other bacteria, viruses, and parasites. The results obtained to date are encouraging, and there is great potential for development of safe, effective, affordable vaccines.     

Immunity induced by live attenuated Salmonella vaccines

Studies on the degree and specificity of protection conferred by immunization with aroA salmonella live vaccines in BALB/c mice are described. Animals were immunized i.v. and challenged orally 3 months later to ensure that the vaccine had been cleared from the tissues. Vaccination with Salmonella typhimurium aroA SL3261 conferred very good protection against virulent S. typhimurium C5 (over 10,000 x LD50). The specificity of cross protection was studied using S. typhimurium, Salmonella enteritidis and Salmonella dublin for vaccination and challenge, including challenge with variants of S. typhimurium and S. enteritidis of similar virulence which differed in the main LPS (lipopolysaccharide) antigen (0-4 or 0-9). S. typhimurium SL3261 gave very good protection against S. typhimurium C5 (0-4), but no protection against S. enteritidis Se795 (0-9). However, challenge with strains differing in the main 0 antigens showed that, although protection was generally better to strains expressing the same LPS type as the vaccine, specificity of protection was determined more by the background (S. typhimurium or S. enteritidis) of the parent strain used for the challenge than by 0 factors 4 or 9, suggesting that other factors could be involved. The nature of the antigen(s) responsible for protection in this model is unclear, but it would not appear to be the main 0-specific antigen. An S. enteritidis Se795 aroA vaccine was far less effective than S. typhimurium SL3261; it conferred good protection against the homologous wild type at 2 weeks post-vaccination, but far less at three months (approx 10-200 x LD50). This was unexpected, as the persistence of the S. enteritidis vaccine in the liver and spleen was similar to that of S. typhimurium SL3261, and the S. enteritidis and S. typhimurium challenge strains were of similar virulence. An S. dublin aroA vaccine conferred similar protection against wild type S. dublin (approx 300 x LD50).     

Oral vaccination against bubonic plague using a live avirulent Yersinia pseudotuberculosis strain

We evaluated the possibility of using Yersinia pseudotuberculosis as a live vaccine against plague because it shares high genetic identity with Y. pestis while being much less virulent, genetically much more stable, and deliverable orally. A total of 41 Y. pseudotuberculosis strains were screened by PCR for the absence of the high pathogenicity island, the superantigens YPM, and the type IV pilus and the presence of the pYV virulence plasmid. One strain (IP32680) fulfilled these criteria. This strain was avirulent in mice upon intragastric or subcutaneous inoculation and persisted for 2 months in the mouse intestine without clinical signs of disease. IP32680 reached the mesenteric lymph nodes, spleen, and liver without causing major histological lesions and was cleared after 13 days. The antibodies produced in vaccinated animals recognized both Y. pseudotuberculosis and Y. pestis antigens efficiently. After a subcutaneous challenge with Y. pestis CO92, bacteria were found in low amounts in the organs and rarely in the blood of vaccinated animals. One oral IP32680 inoculation protected 75% of the mice, and two inoculations induced much higher antibody titers and protected 88% of the mice. Our results thus validate the concept that an attenuated Y. pseudotuberculosis strain can be an efficient, inexpensive, safe, and easy-to-produce live vaccine for oral immunization against bubonic plague.     

The use of two live attenuated vaccines to immunize egg-laying hens against Salmonella enteritidis phage type 4

Laying hens aged 24 weeks were vaccinated twice, separated by a 2-week interval, with spectinomycin-resistant mutants of either the Salmonella gallinarum 9R vaccine or a rough, aroA insertion mutant of a S. enteritidis phage type 4 strain. The 9R strain was given intramuscularly, the rough, aroA mutant both intramuscularly and orally. Two weeks after the second vaccination the immunized and a control group of unimmunized birds were challenged with a nalidixic acid-resistant mutant of a fully virulent phage type 4 strain. Full post-mortem examinations were carried out and eggs were examined bacteriologically for 3 weeks after challenge. Immunization with the 9R strain produced a marked reduction in the number of isolations of the challenge strain from a number of organs, including the ovaries. In contrast, immunization with the rough, aroA mutant produced little change in the isolation rate from the ovaries with small reductions for the liver and spleen. Both candidate vaccines produced a reduction in the number of isolations from laid eggs. The 9R strain was itself isolated from the ovaries throughout the period of examination whereas the aroA strain was not. Sera taken from the birds immediately prior to challenge were examined by slide agglutination using live S. enteritidis cells. Sera from 9R vaccinated birds contained agglutinins whereas sera from most of the chickens immunized with the rough, aroA mutant did not.     

Protection against dengue virus by non-replicating and live attenuated vaccines used together in a prime boost vaccination strategy

A new vaccination strategy for dengue virus (DENV) was evaluated in rhesus macaques by priming with tetravalent purified inactivated virus (TPIV) or tetravalent plasmid DNA vaccines expressing the structural prME gene region (TDNA) then boosting 2 months later with a tetravalent live attenuated virus (TLAV) vaccine. Both vaccine combinations elicited virus neutralizing (N) antibodies. The TPIV/TLAV combination afforded complete protection against DENV 3 challenge at month 8. In a second experiment, priming with TPIV elicited N antibodies against all four serotypes (GMT 1:28 to 1:43). Boosting with TLAV led to an increase in the GMT for each serotype (1:500 to 1:1200 for DENVs 1, 3, and 4, and greater than 1:6000 for DENV 2), which declined by month 8 (GMT 1:62 for DENV 3, 1:154 for DENV 1, 1:174 for DENV 4, and 1:767 for DENV 2). After challenge with each one of the four DENV serotypes, vaccinated animals exhibited no viremia but showed anamnestic antibody responses to the challenge viruses.     

Oral vaccination with an attenuated Salmonella typhimurium strain expressing Borrelia burgdorferi OspA prevents murine Lyme borreliosis.

Borrelia burgdorferi is the causative agent of Lyme disease. In the mouse model, protection is correlated with the development of antibodies to a major outer surface protein, OspA. In this study, we expressed OspA in an attenuated strain of Salmonella typhimurium and tested the efficacy of the transformed strain in protecting against disease. We show that mice inoculated by gavage developed high titers of anti-OspA antibodies and were protected against an intradermal challenge with the spirochete.     

Oral vaccination against Porteus mirabilis.

Mice given a single dose of proteus vaccine orally were protected against 1 MLD (minimum lethal dose) of autologous Proteus mirabilis by the fourth day after vaccination. Three doses of oral vaccine induced protection against 1 MLD autologous challenge for 7 days after vaccination and partial protection for a further 8 weeks. Cross-protection against different strains of Pr. mirabilis and against some strains of Pseudomonas aeruginosa, Serratia marcescens and Providencia species was found in mice immunized orally on three consecutive days with a bivalent proteus vaccine.     

Immunization of mice against Plasmodium vinckei with a combination of attenuated Salmonella typhimurium and malarial antigen.

Infection with the blood stage of the malaria parasite Plasmodium vinckei is uniformly lethal in mice. We found that immunization of BALB/c mice with a combination of killed P. vinckei antigens and an attenuated (aroA) Salmonella typhimurium strain induces high levels of protection against challenge with live P. vinckei. This is especially significant because, in our previous studies, immunization of mice with killed P. vinckei antigens and adjuvants such as Bordetella pertussis, complete Freund adjuvant, and saponin failed to induce protective immunity. Immunization with attenuated S. typhimurium alone did not provide any nonspecific immunity. In vivo depletion of CD4+ T cells in the mice immunized with attenuated S. typhimurium and P. vinckei antigens caused the loss of their immunity. Expression of this immunity required the presence of a spleen. These results support our previous hypothesis that a blood stage malaria vaccine may need both induction of CD4+ T cells specific for the parasite and modification of the spleen with a vaccine vehicle. Therefore, attenuated Salmonella strains such as the one used in this study, when expressing recombinant malarial antigens, might fulfill this requirement.     

Development of a live attenuated vaccine against turkey rhinotracheitis

Three preparations of a strain of turkey rhinotracheitis (TRT) virus were tested for their ability to protect turkey poults against challenge with virulent virus given 3 weeks later. The preparations were as follows: one had been passaged in turkey embryo tracheal organ culture (TOC) 98 times, another had been passaged in primary chick embryo fibroblast (CEF) monolayers 28 times and the third had undergone 17 passages in Vero cell monolayers. Each was administered by the eyedrop route to groups of 21-day-old TRT-seronegative turkey poults. The TOC preparation caused clinical signs consistent with TRT infection, indicating the virus had not been attenuated. The CEF and Vero preparations produced no clinical effects. Following challenge with virulent TRT virus at 21 days post-inoculation, the CEF group developed clinical signs consistent with TRT but the TOC and Vero virus groups showed none. All other parameters correlated with these findings. All groups showed an increase in specific SN and ELISA antibodies following challenge. These results indicated that after relatively few passages in Vero cells, this strain of TRT virus became satisfactorily attenuated and was able to offer protection against clinical disease following experimental challenge. Two of the three virus preparations (TOC and Vero) were also shown to spread from the inoculated birds to uninoculated contact birds, introduced into the groups at 5 days post-inoculation, and they induced protection in these contacts against virulent virus challenge.     

Duration of immunity produced by a live attenuated vaccine against avian pneumovirus type C.

A recently developed live, attenuated vaccine against avian pneumovirus (APV) was found to be safe and protective in experimental birds. Duration of immunity following a single dose of this experimental vaccine in 1-week-old turkey poults is described. Two groups each of 60 poults were housed in separate isolation rooms. Birds in group one were inoculated oculonasally at 1 week of age with the vaccine. The second group served as a non-vaccinated group and was inoculated with mock-infected cell culture fluid. At 3, 7, 10, and 14 weeks post vaccination, 15 birds from each of the groups were removed to separate isolation rooms and challenged with virulent APV. Taken together, data on clinical signs and virus detection in choanal swabs following each challenge indicated that the vaccine was able to protect birds for up to 14 weeks post vaccination. Peak antibody levels were attained 7 weeks post vaccination and declined thereafter. These results indicated that this experimental vaccine induced protection against APV even in the absence of high antibody titres.     

Toxicity of lipopolysaccharide and of soluble extracts of Salmonella typhimurium in mice immunized with a live attenuated aroA salmonella vaccine.

Mice immunized intravenously 10 days earlier (but not those immunized 2 months earlier) with an attenuated Salmonella typhimurium SL3261 aroA live vaccine and tested for delayed-type hypersensitivity by injection of crude Salmonella extracts in the footpad can die within 24 to 48 h of an unexplained allergic reaction. The lethal reaction could be prevented by prior administration of anti-tumor necrosis factor alpha serum. Injection of lipopolysaccharide (LPS) (either purified phenol-water-extracted [Westphal] LPS or protein-rich trichloracetic acid-extracted [Boivin] LPS) was also lethal for mice immunized 10 days before. An LPS-rich crude Salmonella extract was more toxic than one which contained less LPS, suggesting that LPS may have been involved in the lethal reactions to crude antigens. Mild alkaline hydrolysis removes O-linked acyl groups from lipid A and eliminates many toxic effects of LPS; however, both Boivin LPS and Westphal LPS remained toxic for immunized mice after alkaline hydrolysis. In contrast, alkaline hydrolysis of crude whole Salmonella extracts (which caused marked protein degradation) reduced the lethal toxicity of the extracts, especially for an LPS-rich preparation. Mice immunized orally with the live vaccine did not show hypersensitivity to either LPS or crude extracts. The results suggest that the lethal reaction to crude Salmonella antigens in mice immunized 10 days earlier is complex, that tumor necrosis factor alpha is involved, and that allergic reactions to crude antigens (but not to LPS alone) can be reduced by mild alkaline hydrolysis.     

Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for T cells against HIV

An immunization strategy using attenuated bacteria to deliver DNA vaccine plasmids to mucosal sites may induce protective T cell responses against sexual HIV transmission. In a murine intranasal (i.n.) immunization model, we demonstrate that transiently persistent Deltaasd Shigella flexneri strain 15D harboring DNA vaccines induces HIV- and SIV-specific gamma interferon (IFN-gamma) producing CD8+ T cells among splenocytes more efficiently than either a longer persisting DeltaaroD Salmonella typhimurium strain SL7207 or transiently persistent S. typhi strain Ty21a harboring DNA vaccines. Also, the frequency of antigen-specific gamma interferon (IFN-gamma) producing cells induced by Shigella 15D harboring a DNA vaccine were comparable to that induced by intramuscular (i.m.) immunization with purified DNA vaccine. Moreover, the magnitude of mucosal and systemic antigen-specific IgA and IgG responses after immunization were dependent upon the route (i.m. vs. i.n.) of inoculation, with i.n. Shigella 15D DNA vaccines generating higher levels of HIV-specific IgA in vaginal washings than i.m. purified DNA vaccine. Deltaasd S. flexneri is a promising vector for mucosal DNA vaccine immunization against HIV.     

Safety and immune responses to attenuated Salmonella enterica serovar typhi oral live vector vaccines expressing tetanus toxin fragment C

Attenuated Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA was used as a vector to deliver fragment C of tetanus toxin as a single-dose oral tetanus vaccine candidate to elicit protective levels of serum tetanus antitoxin. Twenty-one healthy adult volunteers received doses of 1.6 x 10(7) to 8.2 x 10(9) CFU of one of two strains, CVD 908-htrA(pTETnir15) or CVD 908-htrA(pTETlpp), which contained plasmid-encoded fragment C, with sodium bicarbonate, and the safety and immune responses to serovar Typhi antigens and tetanus toxin were assessed. No volunteer had fever or positive blood cultures after vaccination, although diarrhea occurred in 3 volunteers and vomiting in 2 volunteers within 3 weeks after vaccination. Most volunteers excreted the vaccine strain in the first 72 h after vaccination. Three of nine volunteers who received 10(8) CFU or higher doses of the CVD 908-htrA(pTETlpp) construct developed rises in serum antitoxin antibodies. The serum and cellular immune responses to serovar Typhi antigens were less frequent than those previously observed in volunteers who ingested the parent strain CVD 908-htrA. This study demonstrates that fragment C of tetanus toxin delivered orally to volunteers in an S. Typhi vector can elicit protective levels of serum antitoxin.     

Anti-viral immunity induced by recombinant nucleoprotein of influenza A virus. III. Delivery of recombinant nucleoprotein to the immune system using attenuated Salmonella typhimurium as a live carrier

A plasmid encoding the influenza nucleoprotein gene from A/NT/60/68 virus was transduced into the attenuated Salmonella typhimurium aroA- strain SL3261. The bacterial vector expressing the viral gene product was able to induce both humoral and cell-mediated immune responses to the nucleoprotein antigen. CD4+ virus-specific T cells capable of proliferation were readily induced and, in some circumstances, class II major histocompatibility complex (MHC)-restricted cytotoxicity was detected. However, virus-specific class I MHC-restricted cytotoxic T lymphocytes (CTL) were not detected after such immunization. Mice immunized orally with the nucleoprotein-expressing bacteria mounted a strong anti-viral antibody response and spleen cells from such mice proliferated specifically to virus challenge in vitro, producing interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Orally immunized mice showed significant protection from challenge infection with influenza virus if the mice were also boosted intranasally before infection.     

Clinical and field trials with attenuated Salmonella typhi as live oral vaccines and as "carrier" vaccines

In recent years there has been a resurgence of research to develop new and improved attenuated strains of Salmonella typhi to function as live oral vaccines against typhoid fever and to serve as "carrier" vaccines to express foreign antigens of other pathogens and deliver them to the immune system. Strain Ty21a has served as a prototype in clinical and field trials to identify the optimal formulations and dosage schedules for live vaccines and to quantitate the duration of protection that can be achieved. Clinical trials with three new attenuated S. typhi candidate vaccines, a Vi+ variant of Ty21a, an aroC,aroD double mutant recombinant strain and a cya,crp double mutant, are underway or will be initiated shortly.     

A highly-safe live auxotrophic vaccine protecting against disease caused by non-typhoidal Salmonella Typhimurium in mice

BackgroundSalmonella enterica serovar Typhimurium (S. Typhimurium) has become an important intestinal pathogen worldwide and is responsible for lethal invasive infections in populations at risk. There is at present an unmet need for preventive vaccines.MethodsIRTA GN-3728 genome was sequenced by Illumina and d-glutamate and d-glutamate/d-alanine knockout-auxotrophs were constructed. They were characterized using electron microscopy, growth/viability curves, reversion analysis, and motility/agglutination assays. Their potential as vaccine candidates were explored using two BALB/c mouse models for Salmonella infections: a systemic and an intestinal inflammation. Clinical signs/body weight and survival were monitored, mucosal lactoferrin and specific/cross-reactive IgA/IgG were quantified by enzyme-linked-immunosorbent assays and bacterial shedding/burden in fecal/tissues were evaluated.ResultsThe d-glutamate auxotroph, IRTA ΔmurI, is highly attenuated, immunogenic and fully protective against systemic infection. The IRTA ΔmurI Δalr ΔdadX double auxotroph, constructed to reinforce vaccine safety, showed a higher level of attenuation and was 100% effective against systemic disease. In the intestinal model, it proved to be safe, yielding a low-degree of mucosal inflammation, short-term shedding and undetectable invasiveness in the long-term, while eliciting cross-reactive fecal IgA/serum IgG against clinically relevant multidrug-resistant (MDR) S. Typhimurium strains. It also conferred protection against homologous oral challenge, and protected mice from local and extra-intestinal dissemination caused by one MDR strain responsible for an international outbreak of highly severe human infections. Additionally, oral vaccination promoted extended survival after lethal heterologous infection.ConclusionThis study yielded a very safe S. Typhimurium vaccine candidate that could be further refined for mucosal application against disease in humans.     

Interleukin-12 is critical for induction of nitric oxide-mediated immunosuppression following vaccination of mice with attenuated Salmonella typhimurium.

Studies from our laboratory have shown that infection of mice with an attenuated strain of Salmonella typhimurium causes a marked suppression in the capacity of splenocytes to generate an in vitro plaque-forming cell (PFC) response to sheep erythrocytes. The suppression has been shown to be mediated by mature, adherent macrophages (Mphis) and nonadherent, precursor Mphis. Nitric oxide has been identified as the suppressor factor. The present study investigated the role of interleukin-12 (IL-12) in the generation of nitric oxide-mediated immunosuppression in this model. Salmonella inoculation resulted in marked suppression of PFC responses and high levels of nitrite production. When mice were treated with anti-IL-12 prior to inoculation, nitrite levels in splenocyte cultures were reduced by 75% and the suppression of PFC responses was prevented. The nonadherent splenocyte fraction from Salmonella-inoculated mice, which contains precursor Mphis and is weakly immunosuppressive, was treated with IL-12 in vitro. IL-12 augmented the capacity of this fraction to suppress PFC responses by normal splenocytes in a coculture system. Additionally, IL-12 induced nitrite and gamma interferon (IFN-gamma) production in a dose-dependent manner. Treatment with anti-IFN-gamma blocked nitrite production and suppression, indicating that IFN-gamma is an important intermediary in the pathway of IL-12-induced immunosuppression. These results indicate that IL-12 is critical for the induction of nitric oxide-mediated immunosuppression following S. typhimurium inoculation and, through its ability to stimulate IFN-gamma production, can induce nitric oxide-producing suppressor Mphis.     

Oral vaccination with Salmonella enterica as a cruzipain-DNA delivery system confers protective immunity against Trypanosoma cruzi

To stimulate both local and systemic immune responses against Trypanosoma cruzi, Salmonella enterica serovar Typhimurium aroA was exploited as a DNA delivery system for cruzipain (SCz). In a murine model we compared SCz alone (GI) or coadministered with Salmonella carrying a plasmid encoding granulocyte-macrophage colony-stimulating factor (GII), as well as protocols in which SCz priming was followed by boosting with recombinant cruzipain (rCz) admixed with either CpG-ODN (GIII) or MALP-2, a synthetic derivative of a macrophage-activating lipopeptide of 2 kDa from Mycoplasma fermentans (GIV). The results showed that protocols that included four oral doses of SCz (GI) elicited mainly a mucosal response characterized by immunoglobulin A (IgA) secretion and proliferation of gut-associated lymphoid tissue cells, with weak systemic responses. In contrast, the protocol that included a boost with rCz plus CpG (GIII) triggered stronger systemic responses in terms of Cz-specific serum IgG titers, splenocyte proliferation, gamma interferon (IFN-γ) secretion, and delayed-type hypersensitivity response. Trypomastigote challenge of vaccinated mice resulted in significantly lower levels of parasitemia compared to controls. Protection was abolished by depletion of either CD4⁺ or CD8⁺ T cells. Parasite control was also evident from the reduction of tissue damage, as revealed by histopathologic studies and serum levels of enzymes that are markers of muscle injury in chronic Chagas' disease (i.e., creatine kinase, aspartate aminotransferase, and lactate dehydrogenase). Enhanced release of IFN-γ and interleukin-2 was observed in GI and GII upon restimulation of splenocytes in the nonparasitic phase of infection. Our results indicate that Salmonella-mediated delivery of Cz-DNA by itself promotes the elicitation of an immune response that controls T. cruzi infection, thereby reducing parasite loads and subsequent damage to muscle tissues.     

Salmonella typhi iron uptake mutants are attenuated in mice.

Iron starvation interferes drastically with the multiplication and virulence of Salmonella typhi mutants defective in enterochelin synthesis or enterochelin transport. Growth of these mutants is inhibited in the presence of human sera and unsaturated transferrin and is restored by fully saturated transferrin. The mutants exhibit decreased ability to grow in HeLa cell monolayers and are attenuated in mice. These findings are consistent with the S. typhi enterochelin system playing a role in the pathogenesis of typhoid fever.