Tetanus toxoid loaded nanoparticles from sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide): evaluation of antibody response after oral and nasal application in mice.

PURPOSE: Aim of the study was the evaluation of the potential of novel tetanus toxoid (TT) loaded nanoparticles (NP) for electing an immune response in mice against TT. METHODS: Six week-old female Balb/c mice were immunized by oral (p.o.), nasal (i.n.) and intraperitoneal (i.p.) application of TT NP loaded by adsorption. As polymer a novel polyester, sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide), SB(43)-PVAL-g-PLGA was used. Blood samples were collected 4 and 6 weeks after immunization and assayed for serum IgG- as well as IgA antibody titers by ELISA. NP formulations varying in size and loading were compared to alum adsorbates as well as to TT solutions. RESULTS: Both, p.o. and i.n. administration of TT associated NP increased serum titers up to 3 x 10(3) (IgG) and 2 x 10(3) (IgA). While small NP induced significantly higher titers then larger ones after oral administration, intermediate NP induced antibodies after nasal application. Of the mucosal routes investigated, i.n. seems to be more promising compared to p.o. immunization. CONCLUSIONS: Antigen loaded NP prepared from surface modified polyesters combined with CT show considerable potential as a vaccine delivery system for mucosal immunization. The results warrant further experiments to explore in more detail the potential use of NP as mucosal vaccine delivery system.

Characterization of four members of a multigene family encoding outer membrane proteins of Helicobacter pylori and their potential for vaccination.

In search of protective antigens which can be used in a vaccine to prevent Helicobacter pylori infection, we report on the identification of four genes, hopV, hopW, hopX and hopY, and the characterization of the corresponding proteins which belong to the H. pylori outer membrane protein (Hop) family containing 32 homologous members, some of which were shown to function as porins. Sequence analysis of 16 different H. pylori strains revealed that the proteins HopV, HopW, HopX and HopY are highly conserved. Localization of HopV, HopW, HopX and HopY at the surface of the bacteria was investigated by immunofluorescence. Using a planar lipid bilayer system the proteins HopV and HopX were shown to form pores with single-channel conductances of 1.4 and 3.0 nS, respectively.

Safety and immunogenicity of an intranasal Pseudomonas aeruginosa hybrid outer membrane protein F-I vaccine in human volunteers.

A hybrid protein [Met-Ala-(His)(6) OprF(190-342)-OprI(21-83)] consisting of the mature outer membrane protein I (OprI) and amino acids 190-342 of OprF of Pseudomonas aeruginosa was expressed in Escherichia coli and purified by Ni(2+) chelate-affinity chromatography. After several studies in healthy volunteers, as well as in patients, had proven the tolerability and immunogenicity of the the OprF-OprI vaccine, after intra-muscular application, we developed an emulgel for intranasal immunization. For this purpose we combined a highly concentrated OprF-I with sodium dodecylsulfate as vehicle and the gel matrix natriumlauryl sulfate. After safety and pyrogenicity evaluations in animals, eight healthy adult human volunteers received the OprF-I gel intranasally three times at 2-week intervals. The vaccination was well tolerated and no side effects were observed. An antibody induction (IgG and IgA) could be detected in the sera. These data support continued clinical investigation of the protection against infections in cystic fibrosis patients and patients prone to P. aeruginosa infections.

Immunogenic efficacy of differently produced recombinant vaccines candidates against Pseudomonas aeruginosa infections.

Three different variants of the recombinant hybrid outer membrane protein OprF (aa 190-342)-OprI (aa 21-83) could be obtained in high yield after expression in Escherichia coli. The hybrid protein was modified N terminally, either with a minimal histidine tag or with a homologous sequence of OprF. Both recombinant proteins were purified by nickel chelate affinity chromatography under native and denaturing conditions, and this produced three suitable candidates for a vaccination trial, protein His-F-I, which was purified in its native as well as in its refolded form; and the native purified N terminally extended protein, ex-F-I. In mice, significantly higher antibody titers and survival rates after challenge with Pseudomonas aeruginosa were observed following immunization with protein His-F-I, purified under native conditions.

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

[Diphtheria antitoxin level 2 years after booster vaccination]. / Diphtherie-Antitoxin-Spiegel zwei Jahre nach Auffrischimpfung.

In a prospective, controlled, randomized, multicenter study the immunogenicity of a single (day 0) and two (day 0, 28) booster vaccination against diphtheria were compared in subjects who had received their last diphtheria vaccination more than 10 years ago. Both short-term and long-term immunogenicity was assessed by determining diphtheria antitoxin levels four weeks after vaccination and after one and two years. 102 subjects received the first booster vaccination, and 83 were vaccinated twice. Prior to the first vaccination 27% of the subjects had a diphtheria antitoxin level below 0.01 I.U./ml; after the first booster only 5% were unprotected. The second booster did not show a significant effect, however, in 1 of the 5 subjects who were still unprotected after the first booster the second elicited an antitoxin level of more than 0.01 I.U./ml. After one and two years 7% and 8% of the subjects had diphtheria antitoxin level below 0.01 I.U./ml. A serological effect of a second booster vaccination four weeks after the first one could not be demonstrated neither after one nor after two years.

Conservation, localization and expression of HopZ, a protein involved in adhesion of Helicobacter pylori.

From a sarkosyl-insoluble outer membrane fraction prepared from the Helicobacter pylori strain ATCC 43504, 19 proteins could be sequenced N-terminally by Edman degradation. Oligonucleotides were deduced and used for screening of a genomic library. From the isolated genes, five code for different members of a H.pylori outer membrane protein (Hop) family. Among these, the hopZ gene was characterized in more detail. It encodes a protein which was shown to be located at the bacterial surface by immunofluorescence studies. Sequence analysis of the hopZ gene from 15 different H.pylori strains revealed the existence of two alleles and the possible regulation of hopZ expression by slipped-strand mispairing within a CT dinucleotide repeat motif located in the signal-peptide coding region. Among the different strains, the influence of this region on the expression of HopZ was analyzed on a translational level by western blot analysis of bacterial extracts and immunofluorescence studies on intact cells. The protein is expressed only in those strains in which the number of the CT dinucleotide repeats allow for an open reading frame encoding the complete protein. Addionally the function of HopZ was investigated in an adhesion assay. The wild-type strain ATCC 43504 adhered to human gastric epithel cells whereas a knockout mutant strain showed significantly reduced binding to the cells.

Diphtheria booster vaccination: one or two injections?

In a prospective, controlled, randomized, multicenter study the immunogenicity and tolerance of a single vaccination (day 0) and two (day 0, 28) booster vaccinations against diphtheria were compared in subjects who had received their last diphtheria vaccination more than 10 years ago. 415 subjects received the first booster vaccination, and 203 were vaccinated twice. The geometric mean diphtheria antitoxin concentration after the first booster (day 28) was 2.354 I.U./ml, and after the second booster (day 56) 2.238 I.U./ml. Prior to the first vaccination 48.9% of the subjects had a diphtheria antitoxin level below 0.1 I.U./m; after the first and second boosters 95.4% and 97.5% of subjects, respectively, showed a level of at least 0.1 I.U./ml. A clear serological effect of a second booster 4 weeks after the first one could not be demonstrated.

A recombinant hybrid outer membrane protein for vaccination against Pseudomonas aeruginosa.

Among the numerous targets which can be used for the development of vaccines against Pseudomonas aeruginosa we focused on the outer membrane proteins OprF and OprI. The C-terminal part of OprF from aa 190 to aa 350 was investigated for its conservation and its localization of B-cell epitopes. A hybrid protein which combines the protective epitopes of OprF and OprI was expressed in E. coli and was proven to be highly protective against an intraperitoneal challenge with P. aeruginosa by active immunization of immunocompromised mice as well as by passive immunization of SCID mice with specific antisera. A purification procedure of the N-terminal His-tagged hybrid antigen was established using immobilized-metal-affinity chromatography. To evaluate its safety and immunogenicity the recombinant protein was purified for the immunization of human volunteers. The OprF/OprI hybrid protein is considered to be a candidate for a vaccine against P. aeruginosa.

Safety and immunogenicity of a Pseudomonas aeruginosa hybrid outer membrane protein F-I vaccine in human volunteers.

A hybrid protein [Met-Ala-(His)6OprF190-342-OprI21-83] consisting of the mature outer membrane protein I (OprI) and amino acids 190 to 342 of OprF of Pseudomonas aeruginosa was expressed in Escherichia coli and purified by Ni2+ chelate-affinity chromatography. After safety and pyrogenicity evaluations in animals, four groups of eight adult human volunteers were vaccinated intramuscularly three times at 4-week intervals and revaccinated 6 months later with either 500, 100, 50, or 20 microg of OprF-OprI adsorbed onto A1(OH)3. All vaccinations were well tolerated. After the first vaccination, a significant rise of antibody titers against P. aeruginosa OprF and OprI was measured in volunteers receiving the 100- or the 500-microg dose. After the second vaccination, significant antibody titers were measured for all groups. Elevated antibody titers against OprF and OprI could still be measured 6 months after the third vaccination. The capacity of the elicited antibodies to promote complement binding and opsonization could be demonstrated by a C1q-binding assay and by the in vitro opsonophagocytic uptake of P. aeruginosa bacteria. These data support the continued development of an OprF-OprI vaccine for use in humans.

Immunization of Aotus monkeys with recombinant Plasmodium falciparum hybrid proteins does not reproducibly result in protection from malaria infection.

Plasmodium falciparum antigens SERP, HRPII, MSAI, and 41-3 have shown promise as vaccine components. This study aimed at reproducing and extending previous results using three hybrid molecules. Antibody responses were reproduced in Aotus monkeys, but solid protection from a P. falciparum blood-stage challenge that showed an unintendedly enhanced pathogenicity was not observed.

Serine-stretch protein (SERP) of Plasmodium falciparum corresponds to the exoantigen Ag2, a target of antibodies associated with protection against malaria.

A mixture of Plasmodium falciparum exoantigens inducing lymphocyte activation and cytokine production was shown to contain the malaria vaccine candidate, the serine-stretch protein. This protein was shown serologically to correspond to Ag2, an exoantigen recognized by antibodies linked with protection against malaria. The glycophorin-binding protein, the histidine-rich protein II, the S-antigen, the heat shock protein 70, the ring-infected erythrocyte surface antigen, and the apical membrane antigen-1 were also shown serologically to be present in the mixture of exoantigens.

Conservation of antigen components from two recombinant hybrid proteins protective against malaria.

Recently, we have shown that two hybrid proteins carrying partial sequences of the blood-stage antigens SERP, HRPII, and MSAI from Plasmodium falciparum confer protective immunity on Aotus monkeys against an experimental parasite infection (B. Knapp, E. Hundt, B. Enders, and H. A. Küpper, Infect. Immun. 60:2397-2401, 1992). The malarial components of the hybrid proteins consist of amino acid residues 630 to 892 of SERP, amino acid residues 146 to 260 of MSAI, and the 189 C-terminal residues of HRPII. We have studied the diversity of these protein regions in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from two different areas where malaria is endemic. The gene regions of SERP and MSAI coding for the corresponding sequences of the protective hybrid proteins and the exon II region of the HRPII gene were amplified by polymerase chain reaction and sequenced. All three regions were found to be highly conserved. In the 262-amino-acid fragment of SERP, one single conservative amino acid substitution was found. The exon II region of HRPII showed only a slight variability in number and arrangement of the repeat units. The 115-amino-acid fragment of MSAI which is located within an N-terminal region known to be conserved among different parasite strains was shown to be the most variable among the vaccine components: amino acid substitutions were found in 14 different positions of this MSAI region when both laboratory strains and field isolates were compared.

Comparison of the effects of adjuvants and adjuvant doses on the quantitative and qualitative antibody response to selected antigens in New World squirrel monkeys Saimiri sciureus.

The effect of several adjuvants and of adjuvant doses on the quantitative and qualitative antibody response to tetanus toxoid (TT) and a recombinant herpes simplex virus peptide (HSVgD) was evaluated in the New World monkey Saimiri sciureus. All adjuvant formulations were effective in inducing a strong antibody response to these antigens. The qualitative antibody response, as defined by monoclonal antibodies 3A2/G6 and 4G3/B5, was determined. Only 3A2/G6+ antibodies were induced after immunization with HSVgD irrespective of the adjuvant used and after immunization with TT alone. However, both types of antibodies were induced against TT in the presence of the adjuvants tested. These data suggest that both adjuvant and antigens can intervene in driving the quality of the antibody response induced after immunization. Strong antibody response to TT was induced at any adjuvant dose tested, suggesting that lower doses are as effective as higher doses. These findings can have implications on the design of future trials of vaccine candidate constructs in monkeys and eventually in human beings.

Protection of Aotus monkeys from malaria infection by immunization with recombinant hybrid proteins.

On the basis of investigations of the malarial blood-stage antigens SERP, HRPII, and MSAI from Plasmodium falciparum, we chose two Escherichia coli-expressed hybrid proteins containing selected partial sequences of these antigens. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding P. falciparum polypeptides. In two independent trials with 13 animals, immunization of Aotus monkeys with either of the two hybrid proteins administered in a well-tolerated oil-based formulation protected the animals from an experimental P. falciparum infection.

Strategies for the development of an antimalarial vaccine.

Susceptible Aotus monkeys were immunized with Escherichia coli-derived fusion proteins containing partial sequences of the proteins MSAI, SERP, HRPII and with a group of three recombinant antigens isolated by screening with an antiserum raised against the protective 41 kDa protein band. HRPII, the combination of the fusion proteins of the 41 kDa group and a mixture of two sequences of SERP conferred significant protection against a challenge infection with Plasmodium falciparum blood stages. Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E. coli containing selected partial sequences. In two independent immunization trials it was shown that immunization of Aotus monkeys with either of the two hybrid proteins can protect the animals from an experimental P. falciparum infection.

Protection of Aotus monkeys after immunization with recombinant antigens of Plasmodium falciparum.

The genus Aotus spp. (owl monkey) is one of the WHO recommended experimental models for Plasmodium falciparum blood stage infection, especially relevant for vaccination studies with asexual blood stage antigens of this parasite. For several immunization trials with purified recombinant merozoite/schizont antigens, the susceptible Aotus karyotypes II, III, IV and VI were immunized with Escherichia coli derived fusion proteins containing partial sequences of the proteins MSAI (merozoite surface antigen I), SERP (serine-stretch protein) and HRPII (histidine alanine rich protein II) as well as with a group of recombinant antigens obtained by an antiserum raised against a protective 41 kD protein band. The subcutaneous application (3x) of the antigen preparations was carried out in intact animals followed by splenectomy prior to challenge, in order to increase the susceptibility of the experimental hosts to the parasite. A partial sequence of HRPII, the combination of three different fusion proteins of the 41 kD group and a mixture of two sequences of SERP in the presence of a modified Al(OH)3 adjuvant conferred significant protection against a challenge infection with P. falciparum blood stages (2-5 x 10(6)) i. RBC). Monkeys immunized with the MS2-fusion protein carrying the N-terminal part of the 195 kD precursor of the major merozoite surface antigens induced only marginal protection showing some correlation between antibody titer and degree of parasitaemia. Based on the protective capacity of these recombinant antigens we have expressed two hybrid proteins (MS2/SERP/HRPII and SERP/MSAI/HRPII) in E. coli containing selected partial sequences of SERP, HRPII and MSAI. Antibodies raised against both hybrid proteins in rabbits and Aotus monkeys recognize the corresponding schizont polypeptides. In two independent immunization trials using 13 animals (age 7 months to 3 years) we could show that immunization of Aotus monkeys with either of the two hybrid proteins administered in an oil-based well tolerated formulation protected the animals from a severe experimental P. falciparum (strain Palo Alto) infection.

A Plasmodium falciparum blood stage antigen highly homologous to the glycophorin binding protein GBP.

We have isolated a gene coding for a protein highly homologous to an antigen known as the glycophorin binding protein (GBP) which was therefore called GBPH. The gene consists of 2 exons interrupted by an intron located at a position corresponding to that of the GBP gene. The deduced amino acid sequence of GBPH comprises 427 residues and is characterized by a signal sequence and by an extended repeat region consisting of 8 units of 40 amino acid residues. The comparison of the amino acid sequences of GBPH and GBP reveals an identity of 69%. Antisera raised against a GBPH fragment that carries part of the repetitive region cross-react with GBP (105 kDa) and additionally detect some bands between 40 and 70 kDa, one of which may correspond to GBPH. The genes coding for GBP and GBPH are located on chromosomes 10 and 14, respectively. The GBP gene is transcribed as a highly abundant 6.5 kb mRNA in the blood-stage form, whereas Northern blot analysis using a GBPH specific probe detects 2 less abundant mRNAs of 2.3 kb and 2.7 kb. Southern blot analysis of P. falciparum DNA identifies a third member of the GBP gene family.

Surface expression of malarial antigens in Salmonella typhimurium: induction of serum antibody response upon oral vaccination of mice.

The Escherichia coli OmpA protein can serve as a carrier for the expression of foreign antigens on the surface of gram-negative bacteria. Employing OmpA vectors, immunogenic moieties of the Plasmodium falciparum blood stage antigens SERP and HRPII have been expressed in the attenuated Salmonella typhimurium SR-11 strain. Upon induction, the malaria specific sequences of 189 (HRPII) and 451 (SERP) amino acids, fused into the OmpA protein, have been expressed. By indirect immunofluorescence studies, live bacteria expressing the fusion proteins react anti-SERP and anti-HRPII sera, respectively, indicating that the hybrid OmpA proteins become integrated into the bacterial outer membrane and expose the malarial antigens at the exterior surface. Mice that were immunized orally with S. typhimurium cells expressing HRPII and SERP on their surface show a humoral immune response as determined by the anti-SERP and anti-HRPII IgG and IgM titres. From these experiments it can be concluded that the OmpA surface expression system in combination with established Salmonella vaccine strains can be used to efficiently deliver large antigens to the mucosal immune system.

Demonstration of alternative splicing of a pre-mRNA expressed in the blood stage form of Plasmodium falciparum.

By screening of a lambda gt11 library from Plasmodium falciparum genomic DNA with an antiserum raised against a 41-kDa protein band, which was shown to confer protective immunity to monkeys, the phage clone 41-3 was identified. The entire 41-3 gene was isolated, and its coding regions were determined by amplification and sequencing of 41-3 specific mRNA fragments. The 41-3 gene has a complex structure consisting of nine exons, encoding 375 amino acids in total with a calculated molecular weight of 43,400. Provided that the N-terminal hydrophobic residues function as signal sequence which is cleaved off, the molecular weight of the 41-3 protein decreases to 41,200 and could therefore be considered to be a component of the protective Mr = 41,000 protein band. Indeed, a 41-kDa protein could be detected by Western blot analysis using antisera raised against different recombinant expression products of the 41-3 gene. We furthermore demonstrate an alternative splice process for the mRNA precursor transcribed from the 41-3 gene to yield at least three distinct mRNAs. The major splice product carries all exons E1 to E9, whereas at least two minor 41-3 mRNA species can be identified which show deletions in the region between exons E5 and E7. The possible role of this differential splice process for the parasite is discussed.