Progress 04/01/17 to 03/31/22
Outputs
Target Audience:The scientific community with special interests in enteric bacterial pathogens transmitted through the food chain to humans and means to prevent such transmission by developing vaccines to prevent infection and colonization of farm animals destined to produce products and/or directly to be marketed. Ultimately, the targets are vet biologics companies that would further develop and ultimately market such vaccines. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student and research associate have been trained in vaccine evaluation studies, food safety, microbiology, and basic immunology analyses. How have the results been disseminated to communities of interest?Roy Curtiss gave seminars at Baylor University, Texas Children's Hospital, International Organization for Mycoplasmology Congress, Washington University in St. Louis, University of Connecticut, University of Virginia, University of Nebraska, and University of Florida. He also gave Zoom presentations at the American Association of Avian Pathology and at CRWAD in 2020 and 2021. Graduate student Gary Closs (with Gireesh Rajashekara) gave two presentations. Gary Closs, Jr., Yosra A. Helmy, Dipak Kathayat, Soo-Young Wanda, Roy Curtiss III, Gireesh Rajashekara. Recombinant attenuated Salmonella vaccines reduce Campylobacter colonization and induce IgY antibodies in chickens. CRWAD Dec. 5-8, 2020, Chicago, IL (virtual-oral). Gary Closs, Jr., Yosra A. Helmy, Dipak Kathayat, Vishal Srivastava, Loic Deblais, Roy Curtiss, and Gireesh Rajashekara. Recombinant Attenuated Salmonella Vaccines to Control Campylobacter in Poultry. Edward Hayes Graduate Research Forum. Columbus Ohio. February 28th, 2020. Oral presentation What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Campylobacter and Salmonella are leading food pathogens worldwide and major public health problems. Among 31 major pathogens, they cause 20% of illnesses, 50% of hospitalizations, and 28% of deaths in 9.4 million episodes of foodborne illnesses. Blocking transmission of these two pathogens through the food chain to humans is a high priority. We have designed, constructed, and evaluated two improved Protective Immunity Enhanced Salmonella Vaccine (PIESV) vector strains to deliver some 18 putative C. jejuni protective antigens to orally inoculate newly hatched chicks. Each constructed vaccine strain was characterized for growth, ability to synthesize and deliver the antigens and to maintain complete genetic and phenotypic stability for some 50 generations of growth under permissive conditions. All constructs were completely sensitive to antibiotics used to treat bacterial infections. A total of six clinical trials were conducted to compare different strains of chickens, vaccine doses, and challenge doses with monitoring for decreases in cecal colonization of the challenge strains and immune responses to C. jejuni antigens. In some trials, chicks were vaccinated with combinations of strains to determine whether the benefits were additive and synergistic. Based on the collective studies, we selected five C. jejuni antigens that consistently induced significant levels of protection against C. jejuni colonization levels, although sterile immunity has so far not been achieved. In addition, we designed and constructed a much-improved PIESV strain to induce protective immune responses against Salmonella serotypes. Design and construction of strains to reduce C. jejuni colonization All strains were derived from S. Typhimurium UK-1, which is superior in invasiveness and immunogenicity as a vaccine vector to deliver protective antigens. The constructed strains exhibit regulated delayed attenuation and regulated delayed protective antigen synthesis such that strains exhibit nearly the same abilities as the wild-type virulent parent to colonize internal effector lymphoid tissues before display of attenuation to eliminate occurrence of disease symptoms. The strains also display a regulated delayed lysis in vivo phenotype and thus exhibit biological containment with no persistence in vivo or survival if excreted into the environment. The PIESV strains c12449 and c12539 have these properties and c12539 has the C. jejuni pgl operon inserted into its chromosome and a modified fliC gene with a sequence permitting the attachment of the universal C. jejuni N-glycan antigen. In using c12449, we introduce a low copy number plasmid pG8R201 encoding the C. jejuni pgl operon since many C. jejuni antigens encoded on regulated delayed lysis plasmid vectors contain the D/E X1 N X2 T/S sequences to which the N-glycan is attached as do some of the S. Typhimurium outer membrane proteins. c12449 ?PmurA25::TTaraCParaBADmurA ?asdA27::TTaraCParaBADc2?pmi-2426 D(wza-wcaM)-8 ?relA197::araCParaBADlacI TT DrecF126 DsifA26 ?wbaP45 ?pagL14::TT araC ParaBAD wbaP ?lpxR9 ?pagP8 c12539 ?PmurA25::TTaraCParaBADmurA ?asdA27::TTaraCParaBADc2?pmi-2426 D(wza-wcaM)-8 ?relA197::araCParaBADlacI TT DrecF126 DsifA26 ?wbaP45 ?pagL14::TT araC ParaBAD wbaP ?lpxR9 ?pagP8 ?cysG123::pgl-(gne-plgG)-12 ?fliC180::cj1433gly21-29 C. jejuni putative protective antigens evaluated for induction of immunity to reduce C. jejuni colonization in chickens We screened 18 putative protective C. jejuni antigens to induce immune responses in newly hatched chicks to reduce cecal colonization by a mixture of 5 strains isolated from poultry. Genes for these antigens were present in a vast majority of screened C. jejuni strains and most proteins had signal sequences implying their surface location. Codon-optimized sequences to enhance stability of mRNA and translation efficiency in Salmonella were inserted into either pG8R111 that lacks secretion signals or pG8R114 that possesses the improved bla SS for type 2 secretion. These plasmids were introduced into c12449(pG8R201) and c12539 and evaluated for phenotypic and genetic stability for over 50 generations of growth under permissive conditions. Evaluation of PIESV constructs in newly hatched chicks Six different trials were conducted to evaluate different constructs individually and in combination. We compared using layer versus broiler chicks (no significant differences observed), doses of vaccine construct administered orally at day of age and without or with a booster vaccination some 10 days later and with variation in the timing of administration and doses of the mixture of C. jejuni challenge strains. We also varied the timing of monitoring titers of mucosal and serum antibodies against C. jejuni and the titers of C. jejuni in cecal contents following challenge infections. Trial 1. 8 groups of 17 layer chicks orally vaccinated or unvaccinated and challenged with 8 X 105 CFU of the C. jejuni cocktail mixture (that overwhelmed any immune response). Trial 2. 11 groups of layer chicks orally vaccinated or unvaccinated and challenged with 4 X 104 CFU of the C. jejuni cocktail mixture (still too high). Elevated IgY antibodies were induced against some antigens, but mucosal IgA titers were insignificantly elevated. Trial 3. We compared PIESV constructs with the capability to add the universal C. jejuni N-glycan antigen on C. jejuni and Salmonella antigens. 13 groups of chicks were included with unvaccinated controls and vaccinated and unvaccinated birds were challenged with either 1 X 103 or 1 X104 CFU of the C. jejuni cocktail mixture. The low dose challenge revealed the most significant levels in reduction in cecal colonization. The c12449(pG8R201) constructs gave improved reductions in cecal colonization compared to the c12449 constructs. Trial 4. In this trial, we compared c12449(pG8R201) and c12539 constructs and did two batches with a total of 25 groups, but also varying the time of challenge after vaccination. The best constructs gave similar but variable results when the same antigen was delivered by the two strains. Good IgY and IgA titers were induced, and results were more clear-cut with a challenge dose of 1 X 103 rather than of 1 X 104 CFU of the C. jejuni mixture. Trial 6 evaluated delivery of a mixture of PIESV constructs each delivering a different C. jejuni antigen in comparison to groups of birds vaccinated with a strain delivering a single antigen. The results clearly demonstrated that delivery of multiple C. jejuni antigens gave more significant reductions in cecal colonization by C. jejuni that did delivery of a single antigen. These observations were parallel to observations that indicated higher IgY and IgA titers against C. jejuni antigens in groups vaccinated with mixtures of PIESV strains each delivering a single C. jejuni antigen. Based on these studies, we selected five C. jejuni antigens to use to develop an improved vaccine to reduce C. jejuni colonization of chickens. These antigens include CjaA (encoded on pG8R86), StiK (encoded on pG8R90), FlaA (encoded on pG8R131), PppA (encoded on pG8R281) and ChpA (encoded on pG8R282). Future work to develop a safe efficacious vaccine to reduce C. jejuni colonization of poultry We continue to improve PIESV vector strains and will test with the vector delivering antigens (CjaA, StiK, FlaA, PppA and ChpA). These studies will be more fully described in a continuation application. Design and construction of a strain to reduce infection of poultry by multiple Salmonella serotypes Since the LPS core is structurally identical in all Salmonella serotypes, regulated loss in the ability to synthesize the LPS O-antigen exposes the LPS core in vaccine strains to induce antibodies reactive against all Salmonella. c12704 also induces antibodies against iron-regulated and Mn regulated outer membrane proteins that induce cross-protective immunity against multiple enteric bacteria.
Publications
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Progress 04/01/20 to 03/31/21
Outputs
Target Audience:Poultry veterinarians, poultry farmers, State and Federal regulatory officials, graduate students, food safety researcher. Changes/Problems:Contending with Covid-19 pandemic and unavailability of animal facilities. What opportunities for training and professional development has the project provided?A graduate student and research associate have been trained in vaccine evaluation studies, food safety, microbiology, and basic immunology analyses How have the results been disseminated to communities of interest? Gary Closs, Jr., Yosra A. Helmy, Dipak Kathayat, Soo-Young Wanda, Roy Curtiss III, Gireesh Rajashekara. Recombinant attenuated Salmonella vaccines reduce Campylobacter colonization and induce IgY antibodies in chickens. CRWAD Dec. 5-8, 2020, Chicago, IL (virtual-oral). Gary Closs, Jr., Yosra A. Helmy, Dipak Kathayat, Vishal Srivastava, Loic Deblais, Roy Curtiss, and Gireesh Rajashekara. Recombinant Attenuated Salmonella Vaccines to Control Campylobacter in Poultry. Edward Hayes Graduate Research Forum. Columbus Ohio. February 28th, 2020. Oral presentation What do you plan to do during the next reporting period to accomplish the goals?Conduct two more trials and measure induction of antibodies against C. jejuni.
Impacts
What was accomplished under these goals?
We modified an immunogenic S Typhimurium UK-1 vaccine vector strain that displays regulated delayed attenuation, regulated delayed synthesis of protective antigens and regulated delayed lysis phenotypes by inserting the C jejuni 14-gene pgl operon that encodes enzymes to synthesize the immuno-protective N-glycan attached to C jejuni surface proteins and inserted the D/E-X-N-Y-T/S N-glycosylation sequence in genes encoding surface antigens. This strain was modified to cause in vivo display of conserved cross-reactive antigens for Fe & Mn uptake to yield a vaccine that induces cross protective immunity to most Salmonella serotypes while delivering multiple C jejuni antigens to induce immunity to C jejuni. Trials with chickens were conducted to assess delivery of putative protective C jejuni antigens to decrease colonization by C jejuni. Trial 1: 136 SPF broiler chicks were used and divided into 8 groups (n=17/group). Group1 pG8R86 vaccinated, Group2 pG8R88 vaccinated, Group3 pG8R89 vaccinated, Group4 pG8R90 vaccinated, Group5 pG8R102 vaccinated, Group6 pG8R128 vaccinated, Group7 vaccinated with empty plasmid. All these Groups were challenged with a cocktail of 5 C jejuni strains isolated from chickens. Group8 un-vaccinated and unchallenged. The first trial yielded no significant difference in C jejuni load of vaccinated versus unvaccinated birds; however, we saw promising results that pG8R90 may be efficient at reducing C jejuni load. Seven days post challenge - pG8Rs 128, 86, and 90 yielded the lowest numerical count (~0.86, ~0.62, ~0.5 log respectively) in comparison to the vector control. During the second necropsy, only pG8R90 yielded a noticeable numerical decrease (~0.54). The next trial adjusted the C jejuni challenge dose and added more pG8Rs. Trial 2: 88 SPF broiler chicks were used. Birds were divided into 11 groups (n=8/group): Group 1 pG8R86 vaccinated, Group 2 pG8R88 vaccinated, Group 3 pG8R89 vaccinated, Group 4 pG8R90 vaccinated, Group 5: pG8R102 vaccinated, Group 6: pG8R128 vaccinated, Group 7 pG8R129 vaccinated, Group 8 pG8R 131 vaccinated, Group 9 pG8R132 vaccinated, Group 10 vaccinated with empty plasmid. All these Groups were challenged with the C jejuni cocktail. Group 11 un-vaccinated and unchallenged. For the second trial, we noticed that pG8R90 and 131 significantly (p<0.05) reduced the C jejuni load in comparison to the vector vaccinated group by (~0.6 log) & (~0.62 log). An indirect ELISA protocol was developed and optimized to test the C jejuni specific IgY & IgA antibodies in chicken blood serum. For Trial 1, birds in pG8R86 and pG8R90 showed a significant induction of IgY titers compared to the unvaccinated, unchallenged negative control group at 14 days post challenge. The birds in the vector control group (who were also challenged with C jejuni) did not show this induction This indicated that induction of the pre-challenge titers resulted from vaccination. In the second trial, pG8R90 vaccinated birds showed an induction of pre-challenge IgY titers in comparison to the negative control birds. These results are consistent with previous data suggesting pG8R90 is among the most efficient in reducing C jejuni load. Trial 3: The third trial used vaccine strains enabling C jejunispecific N-glycan recognition sequences for efficient N-glycosylation. The birds were divided into 13 groups, the first 4 groups were vaccinated but not challenged with C jejuni to analyze the impact of vaccinations on young chickens' blood serum antibodies, Groups 5-8 were vaccinated and infected with a lower dosage of C jejuni cocktail at 103 CFU per bird, and Groups 9-12 were vaccinated and infected with a higher dosage of C jejuni cocktail at 104 CFU per bird, group 13 was the un-vaccinated and unchallenged negative control. Group1 pG8R86 vaccinated, Group2 pG8R90+201 vaccinated, Group3 pG8R131 vaccinated, and Group4 vaccinated with empty plasmid were not challenged with the C. jejuni cocktail, Group5 pG8R86 vaccinated (n=20), Group6 pG8R90+201 vaccinated (n=18), Group7 pG8R131 vaccinated (n=20), and Group8 vaccinated with empty plasmid (n=20). All these Groups were challenged with 1x 103 CFU C. jejuni cocktail per bird. Group9 pG8R86 vaccinated and challenged with 1x 104 CFU C. jejuni cocktail per bird, Group10 pG8R 90+201 vaccinated (n=18), Group11 pG8R131 vaccinated (n=20), and Group12 vaccinated with empty plasmid (n=20). All these Groups were challenged with 104 CFU of the C jejuni cocktail per bird. Group13 un-vaccinated & unchallenged (n=18). Half of the remaining birds in the groups were euthanized 9 days post infection (day 27 of study) and the other half 17 days post infection (day 35). Blood was collected from birds pre-C jejuni challenge and at each necropsy to confirm the findings in previous studies that IgY induction is caused by the vaccine constructs and not C jejuni challenge. At 9 days post infection the low dose vaccinated groups showed a significant reduction of C jejuni compared to the Vector control in a two-tailed student T test. The group pG8R86 low dose group showed ~2 log difference, pG8R90+201 Low dose showed ~0.7 log reduction, and pG8R131 showed ~1.4 log reduction. There were no significant results in the high dose groups at 9 days post infection. Similarly at 17 days post infection, pG8R86, pG8R90+201 and pG8R131 low dose groups yielded significant differences at approximately 3.7, 2.4 & 1.7 logs respectively in comparison to the vector control low dose birds. At 17 days post infection the pG8R90+201 high dose group showed a significant log reduction (~1 log) in comparison to the vector control. The results of pG8R90+201 high dose group at 17 days post infection was consistent with Trial 2 where birds received a similar dose of C jejuni but were removed from the study earlier. The results of Trial 3 showed that optimization of the vaccines was effective in reducing C jejuni load demonstrating improved vaccine efficacy. Trial 4: Vaccine groups with an extra plasmid encoding the 12-gene pgl operon in c12449 to compare with c12539, were included to assess which was better at C. jejuni reduction. This was done to further analyze the role of the pgl operon and N-glycosylation in improving vaccine efficacy. 13 groups were included with 20 birds/group. Group1 vaccinated with pG8R86 in c12539, Group2 vaccinated with pG8R90 in c12539, Group3 vaccinated with pG8R131 in c12539, Group4 vaccinated with pG8R281 in c12539, Group5 vaccinated with pG8R282 in c12539, Group6 vaccinated with empty plasmid pG8R111(empty vector) in c12539, Group7 vaccinated with pG8R-86 + pG8R201 in c12449, Group8 vaccinated with pG8R90 + pG8R201 in c12449, Group9 vaccinated with pG8R131 + pG8R201 in c12449, Group10 vaccinated with pG8R281 + pG8R-201 in c12449, Group11 vaccinated with pG8R282 + pG8R201 in c12449 and Group12 vaccinated with empty plasmid pG8R111 + pG8R201 in c12449. All these groups were challenged with the C. jejuni cocktail. Group13 un-vaccinated and unchallenged. In addition to these groups 12 more groups were included, replicating each vaccine group with no challenge (including both empty plasmid controls) and were included for weight analysis. Following the timeline of the previous trial, at 9 days post challenge, c12539 with pG8R282 gave a 2-log C. jejuni reduction followed by pG8R281 and pG8R86 both yielding ~ a 1.4 log reduction, and pG8R131 with ~1 log reduction. In contrast, none of the pG8R201 groups (c12449) showed significant reduction at 9 days but all except pG8R282+201 showed highly significant reduction at 17 days post challenge. At 17 days post challenge, pG8R86+201 had a ~1.8 log, pG8R281+201 yielded a 1.4 log reduction, pG8R90+201 yielded a ~1.3 log reduction and pG8R131+201 yielded a 1 log reduction. Only pG8R281 resulted in significant reduction at 17 days post challenge.
Publications
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Progress 04/01/19 to 03/31/20
Outputs
Target Audience:Poultry veterinarians, poultry farmers, state and federal regulatory officials, graduate students, food safety researchers, manufacturers of vaccines for animal health, academic researchers focused on pathogenicity of pathogens, pathogen transmission and prevention, graduate and undergraduate students enrolled in course of vaccines and vaccinology. Changes/Problems:The recent global pandemic, laboratory shutdown, and animal rooms availability have postponed the schedule of the chicken trials and additional studies at both UF and OSU. However, the OSU has allowed staged research reopening from June 22nd and we have re-initiated our proposed studies in a limited capacity consistent with OSU requirement of 25% occupancy and we are expected to come back to work full time sometime in August if things go well with pandemic. At UF we had a fan failure in our ABSL-2 poultry facility building earlier this year and then the SARS-CoV-2 pandemic shut down research with or without animals. Although lab research resumed June 22nd, we will not set fertile eggs until the week of June 29th. What opportunities for training and professional development has the project provided?A graduate student and research associate have been trained at OSU in vaccine evaluation studies, food safety, microbiology, and basic immunology analyses. At UF, continued research on this project will be the basis for the PhD dissertation research of a DVM graduate student. How have the results been disseminated to communities of interest?The current findings were presented in an oral presentation at the 2020 Edward F. Hayes Graduate Research Forum. The title of the talk was "Recombinant Attenuated Salmonella Vaccines to Control Campylobacter in Poultry" and the PhD student working on the project presented it. Presentations will be made at the forthcoming virtual AAAP and CRWAD meetings. What do you plan to do during the next reporting period to accomplish the goals?At the OSU we plan to run another trial of low-dose challenge Campylobacter using the recent changes to constructs and study design of Trial 3. Additionally, we plan to take blood samples at more time points (pre-vaccination, pre- challenge, and post challenge) during the study to further analyze the role the vaccines play in providing immunogenic protection. We will conduct more immunology studies to analyze the role of IgA antibodies in Trial 2 and subsequent trials. At UF we will be testing our new PIESV vector strains for induction of protective immunity against infection of day-of-hatch chicks with several Salmonella serotypes. These studies will be conducted in conjunction with testing the new SDAAS strains for in ovo inoculation of 18-day embryos to induce innate immunity to enhance the level of protection induced by vaccination of newly hatched chicks with PIESV strains.
Impacts
What was accomplished under these goals?
Studies to prevent C. jejuni colonization in broilers. Previous studies reported last year made use of PIESV vector strain c12452 DPmurA25::TTaraCParaBADmurA DasdA27::TTaraCParaBADc2Dpmi-2426 DwaaL46 DpagL64::TT rhaRS PrhaBAD waaL D(wza-wcaM)-8 DrelA197::araCParaBADlacI TT DrecF126 DsifA26 DompA11 DsopB1925 to deliver C. jejuni antigens specified by codon-optimized gene sequences on pG8R regulated delayed lysis vectors. We report here results of measuring induced antibody titers in broilers vaccinated with these constructs. This year we performed more immunology analyses on previously conducted studies. An indirect ELISA protocol was developed and optimized to test the Campylobacter jejuni specific IgY and IgA antibodies in chicken serum. For Trial 1, birds receiving pG8R86 and pG8R90 showed a significant induction of IgY antibody titers compared to the unvaccinated, unchallenged negative control group at 14 days post challenge. The birds in the vector control group challenged with Campylobacter did not show this induction. There was no significant induction of IgA antibodies 14 days post challenge. Similarly, in the second trial, pG8R90 vaccinated birds showed an induction of pre-challenge IgY titers in comparison to the negative control birds. These results are consistent with the previous microbiological data suggesting pG8R90 is among the most efficient RASVs in reducing campylobacter load. Additionally, we conducted the third trial using the C. jejunispecific N-glycan recognition sequences allowing for efficient N-glycosylation. The birds were divided into 13 groups, the first 4 groups were vaccinated but not challenged with Campylobacter to analyze the impact of vaccinations on young chickens' serum antibodies. Groups 5-8 were vaccinated and infected with a low dose of C. jejuni cocktail at 1x 103 CFU per bird and Groups 9-12 were vaccinated and infected with a high dose of C. jejuni cocktail at 1x 104 CFU per bird. Group 13 is the un-vaccinated and unchallenged negative control. The PIESV vector strain was c12523 ?PmurA25::TTaraCParaBADmurA ?asdA27::TTaraCParaBADc2?pmi-2426 D(wza-wcaM)-8 ?relA197::araCParaBADlacI TT DrecF126 DsifA26 ?wbaP45 ?pagL14::TT araC ParaBAD wbaP ?lpxR9 ?pagP8 ?cysG123::pgl-(gne-plgG)-12. This strain has the C. jejuni pgl operon inserted into the S. Typhimurium chromosome. This operon specifies the enzymes needed for the synthesis of the C. jejuni N-glycan that will be ligated to Salmonella and C. jejuni proteins with the D/E-X1-N-X2 T/S sequence. Based on results from prior vaccination studies, we chose to study delivery of CjaA encoded on pG8R86, antigen cj0998c encoded on pG8R90 and FlaA encoded on pG8R131. We added the pSC101 ori plasmid pG8R201 also encoding the pgl operon to c12523 with pG8R90. Group 1: pG8R86 vaccinated and no infection with C. jejuni cocktail, Group 2: pG8R90+pG8R201 vaccinated and no infection with C. jejuni cocktail, Group 3: pG8R131 vaccinated and no infection with C. jejuni cocktail, Group 4:: vaccinated with empty plasmid and no infection with C. jejuni cocktail, Group 5: pG8R86 vaccinated and challenged with 1x 103 CFU C. jejuni cocktail per bird (n=20), Group 6: pG8R90+pG8R201 vaccinated and challenged with 1x 103 CFU C. jejuni cocktail per bird (n=18), Group 7: pG8R131 vaccinated and challenged with 1x 103 CFU C. jejuni cocktail per bird (n=20), Group 8: vaccinated with empty plasmid challenged with 1x 103 CFU C. jejuni cocktail per bird (n=20), Group 9: pG8R86 vaccinated and challenged with 1x 104 CFU C. jejuni cocktail per bird (n=20), Group 10: pG8R90+pG8R201 vaccinated and challenged with 1x 104CFU C. jejuni cocktail per bird (n=18), Group 11: pG8R131 vaccinated and challenged with 1x 104 CFU C. jejuni cocktail per bird (n=20), Group 12: vaccinated with empty plasmid challenged with 1x 104 CFU C. jejuni cocktail per bird, Group 13: un-vaccinated and unchallenged (n=18). Groups 1-4 analyses have yet to be conducted and are not included in this summary. Half of the birds in the remaining groups were euthanized 9 days post infection (day 27 of study) and the remaining birds 17 days post infection (day 35). Blood was collected pre- Campylobacter challenge and at each necropsy to confirm the findings in previous studies that the IgY induction is caused by the vaccine constructs and not Campylobacter challenge. At 9 days post infection the low dose vaccinated groups showed a significant reduction of Campylobacter compared to the Vector control in a two-tailed student T test. The pG8R86 low-dose group showed ~2 log difference, pG8R90+pG8R201 low-dose showed ~0.7 log reduction, and pG8R131 showed ~1.4 log reduction. There were no significant results in the high dose groups at 9 days post infection. Similarly, at 17 days post infection, pG8R86, pG8R90+pG8R201, and pG8R131 low dose groups yielded significant reductions at approximately 3.7, 2.4, and 1.7 logs respectively in comparison to the vector control low dose birds. At 17 days post infection the pG8R90+201 high dose group showed a significant reduction (~1 log) in comparison to the vector control. The results of pG8R90+pG8R201 high dose group at 17 days post infection was consistent with trial 2 where birds received a similar dose of Campylobacter but were removed from the study earlier. The results of Trial 3 show that the optimized vaccines are effective in reducing Campylobacter load, as observed last year. New PIESV vector strain constructed: To further improve immune responses against the C. jejuni specific N-glycan on all C. jejuni strains, we added a modified FliC subunit with a C-terminal sequence for N-glycan attachment. This modified flagellin is secreted by the flagellar type 3 secretion system. c12539 has the genotype ?PmurA25::TTaraCParaBADmurA ?asdA27::TTaraCParaBADc2?pmi-2426 D(wza-wcaM)-8 ?relA197::araCParaBADlacI TT DrecF126 DsifA26 ?wbaP45 ?pagL14::TT araC ParaBAD wbaP ?lpxR9 ?pagP8 ?cysG123::pgl-(gne-plgG)-12 ?fliC180::cj1433gly21-29. We have introduced pG8R86(CjaA), pG8R131(FlaA), pG8R90(cj0998c), pG8R280(cj0034c), pG8R281(cj0034c with T2SS) and pG8R282(cj0427) into c12539(pG8R201). These constructs have been sent to OSU. Studies to prevent Salmonella colonization in broilers. We conducted studies at UF to evaluate vaccination of day-of-hatch chicks with the PIESV strain c12341 DPmurA25::TTaraCParaBADmurA DasdA27::TTaraCParaBADc2Dpmi-2426 DwaaL46 DpagL64::TT rhaRS PrhaBAD waaL D(wza-wcaM)-8 DrelA197::araCParaBADlacI TT DrecF126 DsifA26 used in our initial studies to deliver C. jejuni antigens, but with disappointing results. The strain also did not induce significant protective immunity to challenge of chicks with the wild-type S. Typhimurium UK-1 c3761. Subsequent work demonstrated that c12341 is very good at stimulating significant innate immune responses but lyses too soon after vaccination to induce long-lasting protective immunities. We used these observations to design superior Self Destructing Attenuated Adjuvant Salmonella (SDAAS) strains that are efficient at inducing innate immune responses that confer significant low-level protection against challenge with a diversity of bacterial, viral and parasite pathogens. We are using SDAAS strains for in ovo administration with no reduction in hatchability to hopefully reduce the ability of Salmonella, C. jejuni and APEC strains from colonizing newly hatched chicks. Going in the opposite direction, we derived a new PIESV strain that persists longer and is expected to induce high-level cellular protective immunity to Salmonella. As a further improvement we made a strain with regulated delayed attenuation due to cessation in synthesis of the Fur and MntR proteins in vivo that induces immune responses against iron and manganese regulated proteins that confer cross-protective immunity against enteric bacteria. We are currently writing a patent application on this improvement.
Publications
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2020
Citation:
Gary Closs, Jr., Yosra A. Helmy, Dipak Kathayat, Vishal Srivastava, Loic Deblais, Anastasia Vlasova, Roy Curtiss, and Gireesh Rajashekara. Recombinant Attenuated Salmonella Vaccines to Control Campylobacter in Poultry and the PhD student working on the project presented it. 2020 Edward F. Hayes Graduate Research Forum, The Ohio State University, Columbus, OH
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Li, P., Q. Liu, H. Luo, K. Liang, Y. Han, K. L. Roland, R. Curtiss 3rd, and Q. Kong. 2018. Bi-valent polysaccharides of Vi capsular and O9 O-antigen in attenuated Salmonella Typhimurium induce strong immune responses against these two antigens. NPJ Vaccines Jan 9;3:1. doi: 10.1038/s41541-017-0041-5. eCollection.
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Progress 04/01/18 to 03/31/19
Outputs
Target Audience:Poultry veterinarians, Poultry farmers, State and Federal regulatory officials, Graduate students, Food safety researchers . The PI has given three seminars at universities and one keynote talk at an international science congress during the past year in addition to one seminar at UF at which efforts to design, construct and evaluate vaccines to enhance food safety were presented and discussed. Changes/Problems:University of Florida : None. Ohio State University: We have faced challenges with infection dose, trying to ensure the birds are properly infected while still using a low dosage for these preliminary studies. Trial 2 has given us more insight on the amount of challenge dose needed to be used in future studies. Additionally, we have made changes to ensure efficient glycosylation of target proteins as many Campylobacter antigens are N-glycosylated. What opportunities for training and professional development has the project provided?Art the University of Florida, Our Brazilian DVM trained in avian medicine in completing work for his MS degree and has been granted a 4 year scholarship to pursue his PhD degree in the College of Veterinary Medicine. Our Venezuelan DVM, also with specialized training in avian medicine and experience in poultry husbandry has joined our group full time to work with our studies in chickens. At Ohio State University, a graduate student and research associate have been trained in vaccine evaluation studies, food safety and microbiology. How have the results been disseminated to communities of interest?Seminars and talks at scientific meetings and conferences. What do you plan to do during the next reporting period to accomplish the goals?We will continue to construct a lysis vector with pSC101 ori derived from pYA4595 with the 11-gene pgl operon (without the pglB gene) to use in place of pG8R201. We will also add the DfliC180::cj1433gly21-29 deletion-fusion mutation as a secreted target to be glycosylated with the C. jejuni N-glycan and also continue to add or modify surface proteins with D/E-X1-N-X2 T/S sequences. We will also use the improved PIESV vector strain c12531 and depending on results might introduce some of the improvements in c12531 into chi12523 or derivatives enabling adding the C. jejuni N-glycan, which we predict will induce immune responses effective against all C. jejuni strain a. Evaluation of PIESV constructs at Ohio State University. Currently we are testing six RASV-Cj strains (pG8R-86, pG8R-88, pG8R-89, pG8R-90, pG8R-102, and pG8R-128) with lysis plasmids in an attempt to determine the levels of reduced C. jejuni colonization and persistence in one-day old chicks. The experiment is being conducted in SPF layer chickens. The chickens were divided into 8 groups (n=17/group): Group 1: pG8R86 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 2: pG8R88 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 3: pG8R89 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 4: pG8R90 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 5: pG8R102 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 6: pG8R128 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 7: Treated with empty plasmid and challenged with cocktail of 5 C. jejuni strains from chicken Group 8: Untreated and unchallenged. The chickens were immunized orally at one- day of age (~200 µL of 10^9 CFU/ chicken in Buffered Saline with Gelatin). The chickens were given a booster after 10 days from the first immunization (Day 11). Chickens will be challenged orally with C. jejuni cocktail strains (~200 µL containing 10^5 CFU of each strain/ chicken in PBS) at 18 days of age. Three chickens will be randomly selected from each group and euthanized for the assessment of the immunologic effect of the vaccine on the chickens prior to challenge. Cecal tonsils will be analyzed for T cells and cytokine response. Serum and intestinal contents will be analyzed for IgY and IgA antibodies. One week following challenge, 7 chickens from group will be sacrificed. Campylobacter load will be assessed in the cecum. Cecal tonsils will analyzed for T cell (CD3+, CD4+, CD8+, CD25+ T cells), and cytokine responses (IL-1beta, IL-8, IFN gamma, IL-10, TNF alpha, TGF beta, TLRs). Serum and intestinal contents will be analyzed for IgY and IgA antibodies. Remaining 7 chickens will be sacrificed 2 weeks following challenge and different tissues will be analyzed as above. We plan to run another trial of low-dose challenge Canpylobacter using the recent changes to constructs The Curtiss lab has made. TheC. jejunispecific N-glycan recognition sequences have been added onto various proteins with D/E X1 N X2 T/S sequences to allow efficient N-glycosylation. The strain has thepgloperon inserted into thecysGgene in the chromosome. Also a low copy number plasmid encoding thispgloperon was added as potential insurance in case the chromosomal insertion is not well expressed. The pG8R90 RASV was the only one stable with this extra plasmid. The pG8R86 vector will also be included since the CjaA sequence contains a D/E X1 N X2 T/S sequence. Future plans: University of Florida Test the remaining three RASV-Cj strains in C. jejuni infected chickens (pG8R-129, pG8R-131, pG8R-132): the experiment will be conducted as mentioned above: Autumn 2018. Test the delivery of multiple RASV. We will use this study to compare the efficacy of individual RASV versus a mixture of RASVs based on the results from above experiment: Winter 2018-2019 Determine if giving a single dose of immunization is satisfactory and if so evaluate the efficacy of lower doses of the aforementioned vaccine: Spring 2019 b. Evaluations at UF. We are currently evaluating immunization of day-of-hatch white layer chicks with one versus two oral doses of one of the PIESV constructs delivering a C. jejuni antigen for ability to protect chicks from colonization after challenge at 3 versus 4 weeks with wild-type S. Enteritidis and S. Typhimurium. This work will be expanded to evaluate challenge with other B, C1, C2, C3, D and E group Salmonella frequently colonizing chickens and passed through the food chain to humans. In addition, day-of-hatch white layer chicks immunized with two doses at 0 and 10 days with a PIESV delivering a C. jejuni antigen are being challenged by the caudal air sac and intratracheal routes with wild-type APEC O1, O2 and O78 strains to examine levels of induced cross-protective immunity to APEC infection. We are monitoring reduction in pathological lesions at autopsy as well as reductions in titers in blood and mortality. As we learn which C. jejuni antigens confer the highest levels of inhibition of colonization by C. jejuni, we will construct lysis plasmid encoding three protective antigens.
Impacts
What was accomplished under these goals?
University of Florida a. Attenuated Salmonella vector strains. We are currently using and evaluating strains with regulated delayed attenuation, regulated delayed protective antigen synthesis and regulated delayed lysis attributes to maximize ability to withstand host defense barriers to ensure efficient invasion and colonization of internal effector lymphatic tissues in chickens. In addition to these means to enhance induction of mucosal and systemic antibody and cellular immune responses, we are evaluating inclusion of other mutations to diminish Salmonella abilities to suppress induction of immunity or to produce immune responses that are subterfuges since they do not contribute to protective immunity. Lastly, we are comparatively evaluating mutational alterations that enhance antibody responses versus cellular immune responses. This is in regard to what immunities best protect against C. jejuni colonization or E. coli APEC infections. Our original PIESV vector strain to deliver C. perfringens antigens that is in process towards obtaining an APHIS license was c12341. Its genotype is listed below. c12341 DPmurA25::TTaraCParaBADmurA DasdA27::TTaraCParaBADc2Dpmi-2426 DwaaL46 DpagL64::TT rhaRS PrhaBAD waaL D(wza-wcaM)-8 DrelA197::araCParaBADlacI TT DrecF126 DsifA26. To deliver C. jejuni protective antigens we made derivatives of c12341 by adding mutations to decrease excess inflammation (DsopB1925) and eliminate a subterfuge (DompA11) illiciting non-protective immune responses. c12417 DPmurA25::TTaraCParaBADmurA DasdA27::TTaraCParaBADc2Dpmi-2426 DwaaL46 DpagL64::TT rhaRS PrhaBAD waaL D(wza-wcaM)-8 DrelA197::araCParaBADlacI TT DrecF126 DsifA26 DompA11 c12452 DPmurA25::TTaraCParaBADmurA DasdA27::TTaraCParaBADc2Dpmi-2426 DwaaL46 DpagL64::TT rhaRS PrhaBAD waaL D(wza-wcaM)-8 DrelA197::araCParaBADlacI TT DrecF126 DsifA26 DompA11 DsopB1925 The ompA gene encodes synthesis of a major outer membrane protein and induces serum antibody titers that represent about half of all serum antibodies to outer membrane proteins. Such anti-OmpA responses confer no protective immunity to Salmonella infection. The SopB protein is immunosuppressive and its absence reduces intestinal inflammation and increases mucosal immune responses. As reported last year, lysis vectors encoding C. jejuni putative protective antigens were introduced into c12452 and the constructs sent to OSU for testing in chickens. Based on these and other results, we have improved the c12341 vector strain to introduce mutations to block metabolism of arabinose and rhamnose, eliminated the Dpmi-2426 mutation and altered the promoter in the rhaRS PrhaBAD promoter to enhance synthesis of the WaaL protein. All of these changes enable the PIESV strain to persist longer by lysing after a few more cell divisions to enhance the levels of induced immune responses. This new vector strain will be used to delivery C. jejuni antigens in evaluation trials this next year. Its genotype is: c12531 DPmurA25::TTaraCParaBADmurA DasdA27::TTaraCParaBADc2 DwaaL46 DpagL38::TT rhaRS PrhaBAD waaL D(wza-wcaM)-8 DrelA197::araCParaBADlacI TT DrecF126 DsifA26 DaraBAD65::TT DrhaBADSR515 b. Regulated lysis plasmid vectors. We have constructed a series of plasmid vectors with differing copy numbers (pSC101 ori, p15A ori and pBR ori) and enabling delivery of synthesized putative protective antigens by lysis of the Salmonella vaccine vector (pG8R111) or in addition by Type 3 secretion (pG8R110) or by a much-improved Type 2 secretion system (pG8R114). We have been using plasmids pG8R111 and pG8R114 (or their precursor parents) with the pBR ori to encode putative C. jejuni protective antigens. c. Recombinant plasmids encoding putative protective C. jejuni antigens for synthesis and delivery by the PIESV vectors. Campy antigens in lysis vectors transformed into c12452 #pG8R Antigen Gene Lysis vector Size bp/aa kDa no BlaSS 86 CjaA cj0982c pG8R17 828/275 31.7 88 Omp18 cj0113 pG8R17 477/158 18.3 89 Peb1 cj0921c pG8R17 744/247 28.4 90 cj0998c pYA4763 552/183 19.6 102 Peb3 cj0289c pG8R17 735/244 28.1 128 Dps cj1534c pYA4763 498/165 18.6 129 TlyA cj0588 pYA4763 810/269 30.6 131 FlaA cj1339c pYA4763 1770/569 60.6 132 PorA/Momp cj1259 pYA4763 1263/420 45.2 pG8R17 is a precursor of the improved pG8R114 and pYA4763 is the precursor of the improved pG8R111. Codon-optimized C. jejuni genes to enhance expression in Salmonella were fused to the improved bla SS in pG8R17 or pG8R114 if they encoded proteins with signal sequences. Codon-optimized sequences for proteins without signal sequences were inserted into pYA4763 or pG8R111. After introduction into the PIESV host, plasmid stability, protein synthesis (by western blot with anti-His serum) and LPS synthesis were validated after 50 generations of growth under permissive conditions. Three new Campylobacter jejuni antigens #pG8R Antigen Gene Signal peptide Lysis vector Size bp/aa Codon optimized kDa 280 Putative periplasmic protein cj0034c 22/23 (euk) pG8R111 own SS 702/233 yes 27.7 281 cj0034c pG8R114 Bla SS 762/253 yes 28.9 282 Hypothetical protein cj0427 none pG8R111 336/111 yes 14.3 d. Evaluation of constructs for inducing immune responses to C. jejuni antigens to also lessen colonization of non-immunized and immunized broiler chicks with C. jejuni. Working out a meaningful means to evaluate vaccine construct efficacy in reducing C. jejuni colonization levels in cecal contents of immunized broiler chicks has not been easy and the literature is full of reports of such difficulties with highly variable results when immunizing with the same C. jejuni antigen but with different challenge strains, breed or strain of chickens and even diet. We have found that low dose challenge reveals the most differences since high challenge doses seem to overwhelm the immunity. We also use a more stringent evaluation in that we use a mixture of C. jejuni strains isolated from chickens and not just the C. jejuni strain used to make the recombinant vaccines. Hopefully, the inclusion of vaccine constructs displaying the C. jejuni N-glycan will confer better levels of protection than seen so far. e. Construction of strains with the pgl operon from Campylobacter that results in N-glycosylation of proteins. 15029 bp of the 12-gene pgl operon (gne, pglKHIJBACDEFG) encoding the ability to synthesize the C. jejuni N-glycan that can attach to surface proteins with the D/E-X1-N-X2 T/S sequences was cloned into pSC101 ori Asd+ vector pYA3337 (expressed from the Ptrc promoter). Also, a plasmid without the pglB gene was constructed (pG8R201). We have now finally succeeded in inserting the pgl operon into cysG gene in the S. Typhimurium chromosome. The resulting strain with its genotype is: c12523 DPmurA25::TTaraCParaBADmurA DasdA27::TTaraCParaBADc2Dpmi-2426 D(wza-wcaM)-8 DrelA197::araCParaBADlacI TT DrecF126 DsifA26 DwbaP45 DpagL14::TT araC ParaBAD wbaP DlpxR9 DpagP8 DcysG123::pgl-(gne-plgG)-12 c12523 has a wild-type waaL gene to enable transport of the N-glycan specified by the pgl operon and its ligation to proteins with the D/E-X1-N-X2 T/S sequence. The strain still exhibits regulated delayed attenuation due to the inability to synthesize and assemplke LPS O-antigen in vivo due to the Dpmi-2426 and DwbaP45 DpagL14::TT araC ParaBAD wbaP mutations. We then introduced the pG8R201 plasmid also encoding the pgl operon and then various regulated lysis plasmids encoding C. jejuni antigens giving the best ability to induce reductions in C. jejuni cecal titers after challenge (see above). Campy antigens in lysis vector in c12523 #pG8R Antigen gene Lysis vector Size/bp supplement needed 86 CjaA cj0982c pG8R17 829 0.1% of L-arabinose and D-mannos 90 + 201 cj0998c pYA4763 552 0.1% of L-arabinose and D-mannose 50 ug/ml Kan 131 FlaA Cj1339c pYA4763 1770 0.1% of L-arabinose and D-mannos Also made c12523 with pYA4763 (empty vector) + pG8R201 as the control.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Burda, W. N., K. E. Brennerman, A. Gonzales, and R. Curtiss III. 2018. Conversion of RpoS- attenuated Salmonella Typhi vaccine strains to RpoS+ improves their resistance to host defense barriers. mSpere Feb 28;3(1). pii: e00006-18. doi: 10.1128/mSphere.00006-18. eCollection 2018 Jan-Feb. PMID: 29507892
Sanapala, S., L. Mosca, S. Wang, and R. Curtiss. 2018. Comparative evaluation of Salmonella Typhimurium vaccines derived from UK-1 and 14028S: Importance of inherant virulence. PloS One Sep 7;13(9):e0203526. doi: 10.1371/journal.pone.0203526. eCollection 2018. PMID: 30192849
Cabello, F. C., S. N. Cohen, R. Curtiss III, G. Dougan, J. van Embden, B. B. Finlay, F. Heffron, D. Helinski, R. Hull, S. Hull, R. Isberg, D. J. Kopecko, S. Levy, J. Mekalanos, J. M. Ortiz, R. Rappuoli, M. C. Roberts, M. So and K. N. Timmis. 2018. Farewell Stan. Environ. Microbiol. 20:2322-2333.
Jiang, Y., Gao, X., K. Xu, J-H. Wang, H-B. Huang, C-W. Shi, W. Yang, Y-H. Kang, R. Curtiss 3rd, G-L. Yang, and C-F. Wang. 2019. A novel Cre recombinase-mediated in vivo minicircle DNA (CRIM) vaccine provides partial protection against Newcastle disease virus. Appl Environ Micro Appl Environ Micro AEM.00407-19. doi: 10.1128/AEM.00407-19. PMID: 31053588
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Progress 04/01/17 to 03/31/18
Outputs
Target Audience:The PI has given some six seminars and keynote talks at universities, science societies and symposia during the past year in addition to two seminars at UF at which efforts to design, construct and evaluate vaccines to enhance food safety were presented and discussed. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?We have taken on a Brazilian DVM trained in avian medicine who has commenced working on this project and who as of May 2018 is now enrolled in the MS degree program in the College of Veterinary Medicine. We just had a Venezuelan DVM, also with specialized training in avian medicine and experience in poultry husbandry join our group as a part-time assistant for our studies in chickens. How have the results been disseminated to communities of interest?Seminars and talks at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?a. Evaluation of PIESV constructs at Ohio State University. Currently we are testing six RASV-Cj strains (pG8R-86, pG8R-88, pG8R-89, pG8R-90, pG8R-102, and pG8R-128) with lysis plasmids in an attempt to determine the levels of reduced C. jejuni colonization and persistence in one-day old chicks. The experiment is being conducted in SPF layer chickens. The chickens were divided into 8 groups (n=17/group): Group 1: pG8R86 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 2: pG8R88 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 3: pG8R89 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 4: pG8R90 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 5: pG8R102 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 6: pG8R128 treated and challenged with cocktail of 5 C. jejuni strains from chickens Group 7: Treated with empty plasmid and challenged with cocktail of 5 C. jejuni strains from chicken Group 8: Untreated and unchallenged. The chickens were immunized orally at one- day of age (~200 µL of 10^9 CFU/ chicken in Buffered Saline with Gelatin). The chickens were given a booster after 10 days from the first immunization (Day 11). Chickens will be challenged orally with C. jejuni cocktail strains (~200 µL containing 10^5 CFU of each strain/ chicken in PBS) at 18 days of age. Three chickens will be randomly selected from each group and euthanized for the assessment of the immunologic effect of the vaccine on the chickens prior to challenge. Cecal tonsils will be analyzed for T cells and cytokine response. Serum and intestinal contents will be analyzed for IgY and IgA antibodies. One week following challenge, 7 chickens from group will be sacrificed. Campylobacter load will be assessed in the cecum. Cecal tonsils will analyzed for T cell (CD3+, CD4+, CD8+, CD25+ T cells), and cytokine responses (IL-1?, IL-8, IFN?, IL-10, TNF?, TGF?, TLRs). Serum and intestinal contents will be analyzed for IgY and IgA antibodies. Remaining 7 chickens will be sacrificed 2 weeks following challenge and different tissues will be analyzed as above. Future plans: Test the remaining three RASV-Cj strains in C. jejuni infected chickens (pG8R-129, pG8R-131, pG8R-132): the experiment will be conducted as mentioned above: Autumn 2018. Test the delivery of multiple RASV. We will use this study to compare the efficacy of individual RASV versus a mixture of RASVs based on the results from above experiment: Winter 2018-2019 Determine if giving a single dose of immunization is satisfactory and if so evaluate the efficacy of lower doses of the aforementioned vaccine: Spring 2019 b. Evaluations at UF. We are currently evaluating immunization of day-of-hatch white layer chicks with one versus two oral doses of one of the PIESV constructs delivering a C. jejuni antigen for ability to protect chicks from colonization after challenge at 3 versus 4 weeks with wild-type S. Enteritidis and S. Typhimurium. This work will be expanded to evaluate challenge with other B, C1, C2, C3, D and E group Salmonella frequently colonizing chickens and passed through the food chain to humans. In addition, day-of-hatch white layer chicks immunized with two doses at 0 and 10 days with a PIESV delivering a C. jejuni antigen are being challenged by the caudal air sac and intratracheal routes with wild-type APEC O1, O2 and O78 strains to examine levels of induced cross-protective immunity to APEC infection. We are monitoring reduction in pathological lesions at autopsy as well as reductions in titers in blood and mortality. As we learn which C. jejuni antigens confer the highest levels of inhibition of colonization by C. jejuni, we will construct lysis plasmid encoding three protective antigens.
Impacts
What was accomplished under these goals?
a. Attenuated Salmonella vector strains. We are currently using and evaluating strains with regulated delayed attenuation, regulated delayed protective antigen synthesis and regulated delayed lysis attributes to maximize ability to withstand host defense barriers to ensure efficient invasion and colonization of internal effector lymphatic tissues in chickens. In addition to these means to enhance induction of mucosal and systemic antibody and cellular immune responses, we are evaluating inclusion of other mutations to diminish Salmonella abilities to suppress induction of immunity or to produce immune responses that are subterfuges since they do not contribute to protective immunity. Lastly, we are comparatively evaluating mutational alterations that enhance antibody responses versus cellular immune responses. This is in regard to what immunities best protect against C. jejuni colonization or E. coli APEC infections. c12341 ?PmurA25::TTaraCParaBADmurA ?asdA27::TTaraCParaBADc2?pmi-2426 ?waaL46 ?pagL64::TT rhaRS PrhaBAD waaL D(wza-wcaM)-8 ?relA197::araCParaBADlacI TT DrecF126 DsifA26 c12417 ?PmurA25::TTaraCParaBADmurA ?asdA27::TTaraCParaBADc2?pmi-2426 ?waaL46 ?pagL64::TT rhaRS PrhaBAD waaL D(wza-wcaM)-8 ?relA197::araCParaBADlacI TT DrecF126 DsifA26 DompA11 c12452 ?PmurA25::TTaraCParaBADmurA ?asdA27::TTaraCParaBADc2?pmi-2426 ?waaL46 ?pagL64::TT rhaRS PrhaBAD waaL D(wza-wcaM)-8 ?relA197::araCParaBADlacI TT DrecF126 DsifA26 DompA11 DsopB1925 c12500 ?PmurA25::TTaraCParaBADmurA ?asdA27::TTaraCParaBADc2?pmi-2426 ?waaL46 ?pagL64::TT rhaRS PrhaBAD waaL D(wza-wcaM)-8 ?relA197::araCParaBADlacI TT DrecF126 SifA+ DompA11 The ompA gene encodes synthesis of a major outer membrane protein and induces serum antibody titers that represent about half of all serum antibodies to outer membrane proteins. Such anti-OmpA responses confer no protective immunity to Salmonella infection. The SopB protein is immunosuppressive and its absence reduces intestinal inflammation and increases mucosal immune responses. The DsifA26 mutation enables Salmonella to escape the Salmonella containing vesicle (SCV, endosome) and this enhances induction of CD8-dependent cellular immunity. The absence of this deletion enhances CD4 responses and antibody production. b. Regulated lysis plasmid vectors. We have constructed a series of plasmid vectors with differing copy numbers (pSC101 ori, p15A ori and pBR ori) and enabling delivery of synthesized putative protective antigens by lysis of the Salmonella vaccine vector (pG8R111) or in addition by Type 3 secretion (pG8R110) or by a much-improved Type 2 secretion system (pG8R114). We have been using plasmids pG8R111 and pG8R114 (or their precursor parents) with the pBR ori to encode putative C. jejuni protective antigens. c. Recombinant plasmids encoding putative protective C. jejuni antigens for synthesis and delivery by the PIRSV vectors. Campy antigens in lysis vectors transformed into c12452 #pG8R Antigen Gene Lysis vector Size bp/aa kDa no BlaSS 86 CjaA cj0982c pG8R17 828/275 31.7 88 Omp18 cj0113 pG8R17 477/158 18.3 89 Peb1 cj0921c pG8R17 744/247 28.4 90 cj0998c pYA4763 552/183 19.6 102 Peb3 cj0289c pG8R17 735/244 28.1 128 Dps cj1534c pYA4763 498/165 18.6 129 TlyA cj0588 pYA4763 810/269 30.6 131 FlaA cj1339c pYA4763 1770/569 60.6 132 PorA/Momp cj1259 pYA4763 1263/420 45.2 pG8R17 is a precursor of the improved pG8R114 and pYA4763 is the precursor of the improved pG8R111. Codon-optimized C. jejuni genes to enhance expression in Salmonella were fused to the improved bla SS in pG8R17 or pG8R114 if they encoded proteins with signal sequences. Codon-optimized sequences for proteins without signal sequences were inserted into pYA4763 or pG8R111. After introduction into the PIESV host, plasmid stability, protein synthesis (by western blot with anti-His serum) and LPS synthesis were validated after 50 generations of growth under permissive conditions. Three new Campylobacter jejuni antigens Antigen Gene Signal peptide Lysis vector Size bp/aa Codon optimized kDa CmeC cj0365c 23/24 (prok) pG8R114 Bla SS 1479/492 ? 54.3 Hypothetical protein cj0427 none pG8R111 336/111 ? 14.3 Putative periplasmic protein cj0034c 22/23 (euk) pG8R111 own SS 702/233 ? 27.7 cj0034c pG8R114 Bla SS 762/253 ? 28.9 pgl operon from Campylobacter 15029 bp of the 12-gene pgl operon (gne, pglKHIJBACDEFG) encoding the ability to synthesize the C. jejuni N-glycan that can attach to surface proteins with the D/E-X1-N-X2 T/S sequences was cloned into pSC101 ori Asd+ vector pYA3337 (expressed from the Ptrc promoter). Also, a plasmid without the pglB gene was constructed (pG8R201). Attempts to insert either operon into cysG gene in the S. Typhimurium chromosome have so far not been successful. Construction of the 11-gene pgl operon (w/o pglB) into pYA4595 (lysis vector with pSC101 ori) is in progress.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
Mellata, M., J. R. Johnson, and R. Curtiss III. 2017. Escherichia coli isolates from commercial chicken meat and eggs cause sepsis, meningitis, and urinary tract infection in rodent models of human infections. Zoonoses Pub. Health 10.1111/zph.12376. PMID: 28703468
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Li, P., Q. Liu, H. Luo, K. Liang, Y. Han, K. L. Roland, R. Curtiss 3rd, and Q. Kong. 2018. Bi-valent polysaccharides of Vi capsular and O9 O-antigen in attenuated Salmonella Typhimurium induce strong immune responses against these two antigens. NPJ Vaccines Jan 9;3:1. doi: 10.1038/s41541-017-0041-5. eCollection.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
Stromberg, Z.R., J. R. Johnson, J. M. Fairbrother, J. Kilbourne, A. Van Goor, R. Curtiss III, and M. Mellata. 2017. Evaluation of Escherichia coli isolates from healthy chickens to determine their potential risk to poultry and human health. PLoS One 12(7):e0180599. doi: 10.1371/journal.pone.0180599. eCollection 2017. PMID: 28671990
- Type:
Journal Articles
Status:
Accepted
Year Published:
2017
Citation:
Laniewski, P., C.-H. Baek, K.L. Roland, and R. Curtiss III. 2017. Analysis of spleen-induced fimbriae production in recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) strains. mBio Aug 22;8(4). pii: e01189-17. doi: 10.1128/mBio.01189-17. PMID: 28830946
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Maddux, J. T., Z. R. Stromberg, R Curtiss 3rd, and M. Mellata. 2018. Recombinant attenuated Salmonella vaccine strain expressing Escherichia coli common pilus has broad protection potential against extraintestinal pathogenic E. coli and Salmonella enterica. Front. Immunol. 8:1280 PMID:29062318
- Type:
Journal Articles
Status:
Accepted
Year Published:
2018
Citation:
Clark-Curtiss, J. E., and R. Curtiss III. 2018. Salmonella vaccines: conduits for protective antigens. J. Immunol. 200:39-48.
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