Immunization of rhesus monkeys with a mucosal prime, parenteral boost strategy protects against infection with Helicobacter pylori.

Rhesus monkeys were immunized with recombinant Helicobacter pylori urease vaccine given solely by the parenteral route or preceded by a priming dose given by the oral route. Two groups of monkeys received parenteral urease with either a synthetic glycolipid adjuvant (Bay) or aluminum hydroxide (alum) as adjuvants. A third group of monkeys received a priming dose of oral urease given with the mucosal adjuvant LT (Escherichia coli heat labile enterotoxin), followed by parenterally administered booster doses of urease adsorbed to alum. Monkeys receiving placebo served as controls. The monkeys received a total of 4 doses of vaccine with the first 3 doses given every 3 weeks and the last booster dose administered 14 weeks later. The monkeys were challenged orally with H. pylori one week after the last vaccine dose and euthanized 10 weeks after challenge, at which time, their stomachs were collected for determination of bacterial colonization and histopathology. Monkeys primed with the oral vaccine and boosted with the parenteral vaccine showed a statistically significant reduction in bacterial colonization when compared to sham-immunized control animals (P = 0.05; Wilcoxon rank sums test). Monkeys receiving parenteral only regimes of urease plus Bay or alum showed no difference in bacterial colonization compared with sham-immunized controls (P = 1.00 and P = 0.33, respectively). The mucosal prime-parenteral boost regime did not cause gastropathy. There was no difference in any of the 3 treatment groups with respect to gastric epithelial changes compared to control animals. There was also no difference in the type and extent of gastric inflammatory cell infiltrates between animals vaccinated by the mucosal prime-parenteral boost strategy and sham immunized controls. However, monkeys receiving the two parenteral-only regimens had slightly elevated gastritis scores.     

Immunization of rhesus monkeys with a mucosal prime, parenteral boost strategy protects against infection with Helicobacter pylori

Rhesus monkeys were immunized with recombinant Helicobacter pylori urease vaccine given solely by the parenteral route or preceded by a priming dose given by the oral route. Two groups of monkeys received parenteral urease with either a synthetic glycolipid adjuvant (Bay) or aluminum hydroxide (alum) as adjuvants. A third group of monkeys received a priming dose of oral urease given with the mucosal adjuvant LT (Escherichia coli heat labile enterotoxin), followed by parenterally administered booster doses of urease adsorbed to alum. Monkeys receiving placebo served as controls. The monkeys received a total of 4 doses of vaccine with the first 3 doses given every 3 weeks and the last booster dose administered 14 weeks later. The monkeys were challenged orally with H. pylori one week after the last vaccine dose and euthanized 10 weeks after challenge, at which time, their stomachs were collected for determination of bacterial colonization and histopathology. Monkeys primed with the oral vaccine and boosted with the parenteral vaccine showed a statistically significant reduction in bacterial colonization when compared to sham-immunized control animals (P=0.05; Wilcoxon rank sums test). Monkeys receiving parenteral only regimes of urease plus Bay or alum showed no difference in bacterial colonization compared with sham-immunized controls (P=1.00 and P=0.33, respectively). The mucosal prime–parenteral boost regime did not cause gastropathy. There was no difference in any of the 3 treatment groups with respect to gastric epithelial changes compared to control animals. There was also no difference in the type and extent of gastric inflammatory cell infiltrates between animals vaccinated by the mucosal prime–parenteral boost strategy and sham immunized controls. However, monkeys receiving the two parenteral-only regimens had slightly elevated gastritis scores.     

Safety and efficacy of E coli enterotoxin adjuvant for urease-based rectal immunization against Helicobacter pylori

Low dose E. coli heat-labile enterotoxin (LT), delivered orally or enterically, has been used as an adjuvant for Helicobacter pylori (H. pylori) urease in healthy adults. In this study we aim to test the safety and adjuvant efficacy of LT delivered rectally together with recombinant H. pylori urease. Eighteen healthy adults without present or past H. pylori infection were enrolled in a double blind, randomized, ascending dose study to receive either urease (60 mg), or urease (60 mg) + LT (5 or 25 microg). The immunization preparation was administered per rectum on days 0, 14 and 28. Serum, stool and saliva anti-urease and anti-LT IgG and IgA antibodies (Abs) were measured and urease-specific and LT-specific antigen secreting cells (ASCs) were counted in peripheral blood at baseline and 7 (ASC counts) or 14 days (antibody levels) after each dosing. Peripheral blood lymphoproliferation assays were also performed at baseline and at the end of the study.Rectally delivered urease and LT were well tolerated. Among the 12 subjects assigned to urease+LT, 2 (16.7%) developed anti-urease IgG Abs, 1 (8.3%) developed anti-urease IgA Abs, and 3 (25%) showed urease-specific IgA(+) ASCs. Immune responses to LT were more vigorous, especially in subjects exposed to 5 microg LT. In the urease+ 5 microg LT group, anti-LT IgG and IgA Abs developed in 60 and 80% of the subjects, respectively, while LT-specific IgG(+) and IgA(+) ASCs were detected in all subjects. The magnitude of the anti-LT response was much higher than the response to urease. No IgA anti-urease or anti-LT Abs were detected in stool or saliva and lymphocyte proliferative responses to urease were unsatisfactory. In conclusion, rectal delivery of 5 microg LT is safe and induces vigorous systemic anti-LT immune responses. Further studies are needed to determine if LT can be an effective adjuvant for rectally delivered antigens.     

Murine antibody response to intranasally administered enterotoxigenic Escherichia coli colonization factor CS6

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of bacterial diarrhea worldwide and is an important cause of infant morbidity and mortality in developing nations. ETEC colonization factors (CF) are virulence determinants that appear to be protective antigens in humans and are the major target of vaccine efforts. One of the most prevalent CF, CS6, is expressed by about 30% of ETEC worldwide. This study was designed to compare the immunogenicity between encapsulated CS6 (CS6-PLG) and unencapsulated CS6. Recombinant CS6 was purified and encapsulated in biodegradable poly(DL-lactide-co-glycolide) (PLG) microspheres using current Good Manufacturing Practices (cGMP). CS6-PLG and CS6 were administered intranasally (IN) to BALB/c mice in three vaccinations 4 weeks apart. Enzyme linked immunosorbent assay (ELISA) was used to measure the anti-CS6 response in serum and mucosal secretions following each of the three inoculations. Mice vaccinated with two or three doses of CS6-PLG demonstrated a significantly greater rise in serum anti-CS6 IgG and mucosal IgA titer values than those immunized with two or three doses of CS6 alone. Three doses of CS6-PLG led to anti-CS6 serum IgG and mucosal IgA titer values 14-fold and 4.4-fold greater, respectively, than three doses of CS6 (P<0.02). IN administered CS6 to mice is safe and highly immunogenic either alone or when encapsulated in microspheres. PLG microsphere encapsulation of CS6 significantly augments the antibody response to that antigen when administered to a mucosal surface.     

Recombinant vaccine against hepatitis E: duration of protective immunity in rhesus macaques

Hepatitis E virus (HEV) is an enterically transmitted virus that causes acute hepatitis. A candidate vaccine containing recombinant HEV capsid protein (56kDa) expressed in insect cells was shown previously to be highly immunogenic when administered in two doses to rhesus monkeys and to protect them from hepatitis E when challenged with a large intravenous dose of homologous or heterologous HEV. In the present study, the effect of a third dose of the same vaccine lot was evaluated and more importantly, the duration of protection following two doses was determined. Rhesus monkeys vaccinated twice with the 56kDa capsid protein were challenged with homologous virus 6 or 12 months after the last vaccination: 3 of 4 monkeys challenged 6 or 12 months later, respectively were protected against viral hepatitis. Similarly, all four of the rhesus monkeys given a third dose of vaccine 1 month prior to challenge were protected against hepatitis. In contrast, all four monkeys given placebo developed hepatitis following challenge. In summary, two doses of HEV vaccine partially protected rhesus monkeys from hepatitis E following intravenous challenge 6 or 12 months after vaccination.     

Helicobacter pylori outer inflammatory protein DNA vaccine-loaded bacterial ghost enhances immune protective efficacy in C57BL/6 mice

Helicobacter pylori (H. pylori) infection is associated with incidents of gastrointestinal diseases in half of the human population. However, management of its infection remains a challenge. Hence, it is necessary to develop an efficient vaccine to fight against this pathogen. In the present study, a novel vaccine based on the production of attenuated Salmonella typhimurium bacterial ghost (SL7207-BG), delivering H. pylori outer inflammatory protein gene (oipA) encoded DNA vaccine was developed, and the efficiency was evaluated in C57BL/6 mice. Significant higher levels of IgG2a/IgG1 antibodies and IFN-γ/IL-4 cytokines were detected after mice were oral administered with oipA DNA vaccine loaded SL7207-BG, indicating that a mixed Th1/Th2 immune response was elicited. When challenged with infective doses H. pylori strain SS1, the ghost based vaccine was capable of reducing bacterium colonization in the vaccinated mice. In addition, codon-optimized oipA plasmid loaded SL7207-BG significantly eliminates H. pylori colonization density in mice model. Thus, it has been demonstrated that this novel bacterial ghost based DNA vaccine could be used as a promising vaccine candidate for the control of H. pylori infection.     

The New World primate, Aotus nancymae, as a model for examining the immunogenicity of a prototype enterotoxigenic Escherichia coli subunit vaccine

The colonization factors (CF) of enterotoxigenic Escherichia coli (ETEC) are being targeted for inclusion in a multi-subunit ETEC vaccine. This study was designed to examine the preclinical safety and immunogenicity of CF CS6, encapsulated in a biodegradable poly(DL-lactide-co-glycolide) (meCS6), and administered in the presence or absence of a mutated heat-labile enterotoxin, LT(R192G), in the non-human primate, Aotus nancymae. A. nancymae were inoculated intranasally (IN) with meCS6 (200 microg; positive control), or intragastrically (IG) with meCS6 (200 or 1000 microg) with or without 2 microg LT(R192G) in three doses given at 2-week intervals. In a second experiment, A. nancymae were inoculated IG with 950 microg of meCS6 with or without 2 microg LT(R192G) in four doses given every 48 h. Blood was collected to assess anti-CS6 and -LT serum immunoglobulin G (IgG) and IgA responses and safety variables (complete blood count and chemistry). Safety parameters were unchanged from baseline following all vaccinations. In Experiment 1, a dose-related serologic response to CS6 was observed; 78.6 and 57.1% of monkeys given 1000 microg meCS6 (n = 14) had a serum IgG and IgA response, respectively, compared to only 28.6% of monkeys given 200 microg meCS6 (n = 14) with a serum IgG and IgA response. No significant effect on the number of responders or the magnitude of responses was observed with the addition of LT(R192G). The three-dose, 2-week regimen with 1000 microg meCS6 was more effective at eliciting an immune response than the four-dose, 48-h regimen with 950 microg meCS6. Results from this study indicate that A. nancymae provide a useful ETEC preclinical safety and immunogenicity model.     

Campylobacter jejuni in broiler chickens: colonization and humoral immunity following oral vaccination and experimental infection

A formalin inactivated, Campylobacter jejuni whole cell vaccine, either with or without Escherichia coli heat labile toxin (LT) as a mucosal adjuvant, was administered orally to broiler chickens. Three vaccine trials were performed, differing in the number of vaccinations, and time of administration, as well as the inclusion and dose of LT. The overall reductions of C. jejuni colonization in the vaccinated chickens ranged from 16 to 93% compared with non-vaccinated controls. Enhanced levels of anti-C. jejuni secretory IgA antibodies were demonstrated in vaccinated chickens. Vaccination also appeared to induce an anamnestic response to C. jejuni antigens in the 14–33 kDa range, as demonstrated by Western immunoblots. Interestingly, the inclusion of LT in the vaccine regimen did not appear to boost the immunogenicity of the vaccine. These results are encouraging and suggest that future development of successful oral vaccines for the control of enteropathogenic Campylobacter in poultry is feasible.     

A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys

A DNA vaccine that expresses the premembrane/membrane (prM) and envelope (E) genes of dengue virus serotype-1 was tested for immunogenicity and protection against dengue-1 virus challenge in Aotus nancymae monkeys. The vaccine, in 1 mg doses, was administered intradermally (i.d.) to three monkeys and intramuscularly (i.m.) to three others. For controls, a 1 mg dose of vector DNA was administered i.d. to two monkeys and i.m. to one. All animals were primed and then boosted at one and five months post priming. Sera were collected monthly and analyzed for dengue-1 antibodies by enzyme linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). Dengue-1 antibodies were detectable in the sera from i.d. and i.m. vaccine inoculated animals one month after the first boost and peaked one month after the second boost. The antibody levels from sera of animals that received the vaccine via the i.d. route were twice those from sera of animals that received the vaccine via the i.m. route. Six months after the second boost all inoculated and two naive monkeys were challenged with 1.25x10(4) plaque forming units (PFU) of dengue-1 virus. Two vaccine immunized animals were protected from viremia while the others showed a reduction in viremia. The mean days of viremia were 1 and 1.3 for the animals that were immunized with the vaccine via the i.d. or i.m. route, respectively vs 4 and 2 mean days of viremia in the animals inoculated with control DNA. Naive animals were viremic for an average of 4 days. All of the three control monkeys that received control DNA inoculum by either the i.d. or i.m. route had an intermittent viremia pattern with one or more negative days interspersed between the positive days. This pattern was not observed in any of the vaccine recipients or the na?ve control monkeys. These results demonstrate that DNA immunization is a promising approach for the development of dengue vaccines and that A. nancymae monkeys are suitable for dengue vaccine trials.     

Immunogenicity and efficacy comparison of MDCK cell-based and egg-based live attenuated influenza vaccines of H5 and H7 subtypes in ferrets

During a pandemic, the availability of specific pathogen free chicken eggs is a major bottleneck for up-scaling response to the demand for influenza vaccine. This has led us to explore the use of Madin-Darby Canine Kidney (MDCK) cells for the manufacture of live attenuated influenza vaccine (LAIV) that provides production flexibility and speed. The present study reports the comparison of the immunogenicity and efficacy of two MDCK-based LAIVs against two egg-based LAIVs prepared from the same pandemic potential strains of H5 and H7 subtypes after a single dose of the vaccine followed by a challenge with a homologous wild type strain. The vaccine strains have been generated by classical method of reassortment using the A/Leningrad/134/17/57 master donor strain. Additionally, a prime-boost regimen of the MDCK-based vaccine followed by a challenge with a homologous wild type strain for H5 and H7 immunized ferrets and also a heterologous wild type strain for the H5 immunized animals was studied. No difference in the hemagglutination inhibition and virus neutralization antibody titers against the homologous virus was observed following a single dose of either egg-based or MDCK-based H5 and H7 LAIV vaccine. A second dose of MDCK-based vaccine significantly boosted antibody titers in the vaccinated animals. Both a single dose or two doses of LAIV provided complete protection from lower respiratory tract infection and resulted in a significant reduction in the virus titers recovered from the throat, nasal turbinates and lungs after challenge with the homologous wild type strain. Protection from a challenge with a heterologous strain of H5 was also observed after two doses of the MDCK-based LAIVs. This data strongly supports the use of MDCK as a substrate for the manufacture of LAIV which ensures reliable quality, safety, production flexibility, speed and breadth of protection, features that are highly critical during a pandemic.     

Evaluation of replication, immunogenicity and protective efficacy of a live attenuated cold-adapted pandemic H1N1 influenza virus vaccine in non-human primates

We studied the replication of influenza A/California/07/09 (H1N1) wild type (CA09wt) virus in two non-human primate species and used one of these models to evaluate the immunogenicity and protective efficacy of a live attenuated cold-adapted vaccine, which contains the hemagglutinin and neuraminidase from the H1N1 wild type (wt) virus and six internal protein gene segments of the A/Ann Arbor/6/60 cold-adapted (ca) master donor virus. We infected African green monkeys (AGMs) and rhesus macaques with 2×10(6) TCID(50) of CA09wt and CA09ca influenza viruses. The virus CA09wt replicated in the upper respiratory tract of all animals but the titers in upper respiratory tract tissues of rhesus macaques were significant higher than in AGMs (mean peak titers 10(4.5) TCID(50)/g and 10(2.0) TCID(50)/g on days 4 and 2 post-infection, respectively; p<0.01). Virus replication was observed in the lungs of all rhesus macaques (10(2.0)-10(5.4) TCID(50)/g) whereas only 2 out of 4 AGMs had virus recovered from the lungs (10(2.5)-10(3.5) TCID(50)/g). The CA09ca vaccine virus was attenuated and highly restricted in replication in both AGMs and rhesus macaques. We evaluated the immunogenicity and protective efficacy of the CA09ca vaccine in rhesus macaques because CA09wt virus replicated more efficiently in this species. One or two doses of vaccine were administered intranasally and intratracheally to rhesus macaques. For the two-dose group, the vaccine was administered 4-weeks apart. Immunogenicity was assessed by measuring hemagglutination-inhibiting (HAI) antibodies in the serum and specific IgA antibodies to CA09wt virus in the nasal wash. One or two doses of the vaccine elicited a significant rise in HAI titers (range 40-320). Two doses of CA09ca elicited higher pH1N1-specific IgA titers than in the mock-immunized group (p<0.01). Vaccine efficacy was assessed by comparing titers of CA09wt challenge virus in the respiratory tract of mock-immunized and CA09ca vaccinated monkeys. Significantly lower virus titers were observed in the lungs of vaccinated animals than mock-immunized animals (p≤0.01). Our results demonstrate that AGMs and rhesus macaques support the replication of pandemic H1N1 influenza virus to different degrees and a cold-adapted pH1N1 vaccine elicits protective immunity against pH1N1 virus infection in rhesus macaques.     

Experiments on monkeys with cholera toxin partially purified and detoxified with formol and glycine

Cholera toxoid partially purified and detoxified with formol and glycine and inoculated in monkeys proved safe. Histological examination revealed no changes in the skin of the animals treated. Blood samples taken from monkeys immunized with 2 doses of toxoid 12 days after the second inoculation revealed appreciable levels of antitoxin in the animals that had received two 100-μg doses of toxoid. The monkeys vaccinated with toxoid, when inoculated with challenge doses of cholera toxin, had the capacity to neutralize the toxin. Detoxification with formol and glycine may be the first step towards the preparation of a highly purified, innocuous, and antigenic cholera toxoid.     

Effectiveness and safety of mutant Escherichia coli heat-labile enterotoxin (LT H44A) as an adjuvant for nasal influenza vaccine

The effectiveness and safety of mutant Escherichia coli heat-labile enterotoxin, LT H44A (His to Arg substitution at position 44 from the N-terminus of the A1 fragment of the A subunit) as an adjuvant for nasal influenza vaccine were examined. (1) When 0.2 microg of LT H44A, together with 0.2 microg of influenza A/PR/8/34 virus (PR8, H1N1) vaccine, was administered intranasally into BALB/c mice (twice, 4 weeks apart), anti-PR8 hemagglutinin (HA) IgA and IgG antibody (Ab) responses were induced at levels that were sufficient to provide either complete protection against infection with a small volume of PR8 virus suspension or partial protection against infection with a lethal dose of the suspension. The dose of the mutant LT and vaccine used here (0.2 microg/ 20 g doses mouse) corresponded to the estimated dose per person, i.e. 0.1 mg/10 kg body weight. (2) Using these vaccination conditions, no additional total IgE Ab responses were induced. (3) The mutant was confirmed to be less toxic than the native LT when the toxicity was analyzed either using Y1 adrenal cells in vitro (1/483 EC(50)) or by an ileal loop test. (4) One hundred micrograms of the mutant, administered intranasally or intraperitoneally into guinea-pigs (Heartley strain, 0.3-0.4 kg), caused no body-weight changes 7 days after administration, although 100 microg of the native LT administered intraperitoneally caused death in all guinea-pigs due to diarrhea within 2 days. The intranasal administration of 100 microg of the mutant resulted in almost no pathological changes in the nasal mucosa 3 days after administration. These results suggest that LT H44A, which can be produced in high yields in an E. coli culture (about 5 mg/l), could be used as one of the effective and safe adjuvants for nasal influenza vaccine in humans.     

U-Omp19 from Brucella abortus increases dmLT immunogenicity and improves protection against Escherichia coli heat-labile toxin (LT) oral challenge

Acute diarrhea disease caused by bacterial infections is a major global health problem. Enterotoxigenic Escherichia coli (ETEC) is one of the top causes of diarrhea-associated morbidity and mortality in young children and travelers to low-income countries. There are currently no licensed vaccines for ETEC. Induction of immunity at the site of entry of the bacteria is key to prevent infection. Current approaches to ETEC vaccines include a less toxic mutant form of E. coli heat-labile toxin (double-mutant heat-labile enterotoxin -dmLT-) with both antigenic and immunostimulatory properties. U-Omp19 is a protease inhibitor from Brucella spp. with immunostimulatory properties that has been used as oral adjuvant. In this work, we use U-Omp19 as adjuvant in an oral vaccine formulation against ETEC containing dmLT in outbred and inbred mice. To evaluate antigen dose sparing by U-Omp19 three different immunization protocols with three different doses of dmLT were evaluated. We demonstrated that U-Omp19 co-delivery increases anti-LT IgA in feces using a mid-dose of dmLT following a prime-boost protocol (after one or two boosts). Oral immunization with U-Omp19 induced protection against LT challenge when co-formulated with dmLT in CD-1 and BALB/c mice. Indeed, there was a significant increase in anti-LT IgG and IgA avidity after a single oral administration of dmLT plus U-Omp19 in comparison with dmLT delivered alone. Interestingly, sera from dmLT plus U-Omp19 vaccinated mice significantly neutralize LT effect on intestine inflammation in vivo compared with sera from the group immunized with dmLT alone. These results demonstrate the adjuvant capacity of U-Omp19 to increase dmLT immunogenicity by the oral route and support its use in an oral subunit vaccine formulation against ETEC.     

Evaluation of the immunogenicity and protective efficacy of a candidate parainfluenza virus type 3 subunit vaccine in cotton rats.

A parainfluenza virus type 3 (PIV3) subunit vaccine consisting of detergent-solubilized, affinity-purified haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins was tested in cotton rats for immunogenicity, short-term effects on virus-induced immunopathology and protective efficacy. Groups of animals were immunized twice, 4 weeks apart, with graded doses of vaccine administered either alone or with aluminium phosphate (AlPO4). The minimum immunogenic dose of vaccine was 0.1 microgram HN and F when the vaccine was given alone and 0.01 microgram when the vaccine was administered with AlPO4 adjuvant. Antibody responses in animals immunized with 1 microgram HN and F mixed with adjuvant were similar to those in control animals infected with live PIV3 intranasally. Pulmonary and nasal wash PIV3 titres generally were inversely correlated with serum antibody levels. Virus titres were significantly reduced in all groups of animals immunized with greater than or equal to 0.1 microgram HN and F compared with control animals immunized with vehicle only. Four days after virus challenge, there was no evidence of enhanced histopathology in lung sections from animals immunized with the candidate vaccine.     

B cell responses in gastric antrum and duodenum following oral inactivated Helicobacter pylori whole cell (HWC) vaccine and LT(R192G) in H pylori seronegative individuals

To investigate whether B cell-specific responses could be elicited in the gastric mucosa of Helicobacter pylori (HP) naive subjects, five volunteers ingested three doses of a HP killed whole cell (HWC) vaccine with 25 microg of recombinant heat-labile toxin (LT(R192G)). Two of three subjects had detectable LT(R192G) and HWC IgA antibody secreting cell (ASC) gastric responses. LT(R192G) and HWC responses in duodenal were 5-14-fold higher than those detected in antral biopsies (P<0.01 and P=0.05, respectively). These results provide the first evidence that specific gastric B cell responses can be induced in HP-non-infected individuals following oral immunization.     

Class-specific antibody responses in school children vaccinated with an A/Brazil/11/78 (H1N1)-like recombinant influenza virus prepared from the A/Leningrad/134/57 paediatric cold-adapted donor strain

Forty-three school children from 8 to 11 years old were vaccinated intranasally with two doses of a paediatric attenuated influenza vaccine developed by reassortment between cold-adapted A/Leningrad/134/57(H2N2) and an A/Brazil/11/78(H1N1)-like strain. Two vaccine doses were administered 1 month apart in a randomized, blind, placebo-controlled study. Although the first vaccine dose had a low infectivity titre, overall 65% of children who received two doses of vaccine showed serological evidence of infection by HI tests. Serum IgA antibody responses against the vaccine strain were detected in nearly 50% of the vaccines and serum IgG antibody responses were detected in approximately equal to 40% by an enzyme immunoassay.     

Immune responses in mice to intranasal and intracutaneous administration of a DNA vaccine encoding Helicobacter pylori-catalase

We previously reported that the intracutaneous injection of DNA vaccines encoding Helicobacter pylori heat shock proteins elicited specific immune responses, and led to reduced infection in mice. In this study, we constructed DNA vaccine encoding H. pylori-catalase (pcDNA3.1-kat) and investigated the immune responses to intranasal and intracutaneous administration of pcDNA3.1-kat. C57/BL6 mice were immunized intracutaneously with 10 microg of pcDNA3.1-kat or intranasally with 50 microg of pcDNA3.1-kat. Catalase-specific IgG antibody was detected in the sera of intranasal and intracutaneous immunized mice. Both intranasal and intracutaneous immunized mice were significantly protected from colonization by H. pylori and had significantly reduced degrees of gastritis. These results demonstrate that DNA vaccine encoding H. pylori-catalase can induce an immune response against H. pylori, and that intranasal immunization works as well as intracutaneous immunization.     

Chimeric flagellin as the self-adjuvanting antigen for the activation of immune response against Helicobacter pylori

Helicobacter pylori infection can cause gastritis, peptic ulcer and can lead to gastric cancer. Lengthy antibiotic therapy does not protect the host against reinfection. H. pylori evolved to evade the recognition of the immune response by modifying several of its components whose orthologous proteins from other bacteria activate the innate immune response. Flagella are essential for the H. pylori effective colonization of human duodenum and stomach. TLR5, a member of the Toll-like receptor family, recognizes flagellin of most bacteria, such as Escherichia coli, but does not recognize the flagellin FlaA of H. pylori. We restored the ability of FlaA for the recognition by TLR5 by engineering a chimeric flagellin, in which both terminal segments of H. pylori flagellin were replaced by the corresponding segments from TLR5-activating E. coli flagellin. Recombinant chimeric flagellin folded correctly and was able to activate TLR5. Significantly increased serum IgG and IgA antibody responses were determined in mice vaccinated with chimeric flagellin in comparison to mice vaccinated with a control protein (FlaA) or negative control. Antibody titers remained high even 8 months after the last immunization. Antibodies were able to bind native flagellin from H. pylori lysate. Vaccination with chimeric flagellin provided mice with significant protection against H. pylori. The approach of chimeric flagellin can therefore generate effective immunogens that enable activation of innate and adaptive immune response and can be used to construct efficient vaccines against H. pylori or other flagellated bacteria that evade TLR5 recognition.     

One or two dose regimen of the SARS-CoV-2 synthetic DNA vaccine INO-4800 protects against respiratory tract disease burden in nonhuman primate challenge model

Safe and effective vaccines will provide essential medical countermeasures to tackle the COVID-19 pandemic. Here, we assessed the safety, immunogenicity and efficacy of the intradermal delivery of INO-4800, a synthetic DNA vaccine candidate encoding the SARS-CoV-2 spike protein in the rhesus macaque model. Single and 2 dose vaccination regimens were evaluated. Vaccination induced both binding and neutralizing antibodies, along with IFN-γ-producing T cells against SARS-CoV-2. Upon administration of a high viral dose (5 × 10⁶ pfu) via the intranasal and intratracheal routes we observed significantly reduced virus load in the lung and throat, in the vaccinated animals compared to controls. 2 doses of INO-4800 was associated with more robust vaccine-induced immune responses and improved viral protection. Importantly, histopathological examination of lung tissue provided no indication of vaccine-enhanced disease following SARS-CoV-2 challenge in INO-4800 immunized animals. This vaccine candidate is currently under clinical evaluation as a 2 dose regimen.