A novel microencapsulated peptide vaccine against hepatitis B.

A 48 amino acid synthetic peptide (S121/48) representing residues 121-167 of the major envelope protein of hepatitis B virus (HBsAg) was successfully encapsulated into polylactide co-glycolide microspheres. A single immunization of the microspheres in BALB/c (H-2d) mice resulted in the production of high-titre anti-HBs antibodies (IgG1-type). The response was long lasting and was superior to that obtained using the same peptide adjuvanted with Freund's complete adjuvant. A T-cell memory response was detected 10 weeks after a booster immunization (approximately 35 weeks after initial immunization) as measured by in-vitro re-stimulation of splenocytes. This study illustrates the feasibility of a single dose vaccine for hepatitis B and is, to our knowledge, the first demonstration of a synthetic peptide immunogen inducing anti-native protein antibodies of comparable titre to those obtained with conventional vaccines for hepatitis B. The suitability of a synthetic peptide vaccine for hepatitis B is discussed.

Formulation of poly(D,L-lactic-co-glycolic acid) microparticles for rapid plasmid DNA delivery.

An optimised water-in-oil-in-water double emulsion process for the microencapsulation of plasmid DNA in poly(D,L-lactic-co-glycolic acid) (PLGA) was used to prepare microparticles from a range of different PLGA formulations. This process has been developed using pharmaceutically accepted solvents and is potentially scaleable. Incorporation of plasmid DNA in the microparticles of up to 11 microg/mg was obtained and the retention of plasmid DNA integrity was considerably greater than previously reported. Microparticle structure was determined, by scanning electron microscopy, to be hollow and size distribution characteristics were found to be independent of polymer formulation. The ability to vary the plasmid DNA release profile by changing the PLGA formulation and polymer concentration used in the encapsulation process was also demonstrated. This ability to control the release profile of the microparticles was shown to be especially important as the physical integrity of the encapsulated plasmid DNA was found to deteriorate with extended release times in vitro.

Immune responses and protection obtained by oral immunization with rotavirus VP4 and VP7 DNA vaccines encapsulated in microparticles.

Protective immune responses in mice were obtained after oral immunization with rotavirus DNA vaccines encapsulated in poly(lactide-co-glycolide) (PLG) microparticles. The DNA vaccines used encoded outer capsid proteins VP4 and VP7; proteins that are the basis for rotavirus serotyping and the generation of virus neutralizing antibodies. One dose of vaccine was given to BALB/c mice by oral gavage (75 microg DNA/mouse). Rotavirus-specific serum antibodies and intestinal IgA antibodies were detectable by 6 weeks postimmunization. After challenge with homologous murine rotavirus at 12 weeks postimmunization, fecal rotavirus antigen was reduced significantly in immunized mice compared with controls. Protective immunity also was generated by oral delivery of unencapsulated VP 7 DNA vaccine but to a lesser degree. These results demonstrate that the oral route is effective for generating protective immune responses with rotavirus DNA vaccines targeting neutralization antigens.

Comparison of the immunological and protective responses elicited by microencapsulated formulations of the F1 antigen from Yersinia pestis.

Purified native F1 antigen from Yersinia pestis was used to assess controlled-release vaccine delivery systems in poly(lactide-co-glycolide) (PLG) microparticles and liposomes. Antigen encapsulated in PLG microparticles induced high serum titres when injected i.p. in mice: mucosal IgA was also detected. Mice immunized with F1 in Alhydrogel or PLGs were protected against subcutaneous challenge with Y. pestis. F1 antigen surface-labelled onto liposome vesicles stimulated high serum titres in Balb/c mice and also induced a mucosal response: F1-labelled liposomes protected mice against challenge with up to 1 x 10(5) organisms. These findings indicate that a significant immune response is induced by immunizing with F1 formulated in PLGs and liposomes and that protection was achieved after only one dose.

Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles.

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.

Oral delivery of micro-encapsulated DNA vaccines.

Oral delivery of vaccines is an attractive alternative to injection. It is a non-invasive procedure which allows access to the gut-associated lymphoid tissues (GALT). Immunisation at GALT results in mucosal immune responses, which may be of particular importance in protection against infection at mucosal surfaces, as well as systemic immune responses. Vaccine antigens can be protected in the gut by encapsulation in poly(DL-lactide-co-glycolide) (PLG) microparticles. Their uptake into the immune inductive tissues of the GALT is mediated by M cells, which selectively phagocytose particles less than 10 microns in diameter. We have developed a method for the PLG encapsulation of plasmid DNA. Encapsulated DNA, expressing the insect protein luciferase under the transcriptional control of the human cytomegalovirus immediate early promoter, was administered to mice by intraperitoneal injection or oral gavage. Intraperitoneal injection of encapsulated DNA elicited good serum IgG and IgM responses and a modest IgA response. Oral administration stimulated good serum antibody titres in all three classes, and in addition, significant levels of mucosal IgA. PLG encapsulation thus has the ability to protect plasmid DNA against degradation after administration, and to facilitate its uptake into appropriate cells for the subsequent expression and presentation of antigen, in such a way as to elicit both systemic and mucosal antibody responses. This may have major implications for the design of novel vaccines and delivery strategies.

Poly(DL-lactide-co-glycolide)-encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration.

We have developed a method for the encapsulation of plasmid DNA in poly(DL-lactide-co-glycolide) microparticles. Encapsulated DNA, expressing the insect protein luciferase under the transcriptional control of the human cytomegalovirus immediate early promoter, was administered to mice by intraperitoneal injection or oral gavage. Intraperitoneal injection of encapsulated DNA elicited good serum IgG and IgM responses, and a modest IgA response. Oral administration stimulated good serum antibody responses in all three classes, and in addition, significant levels of mucosal IgA. PLG encapsulation thus has the ability to protect plasmid DNA against degradation after administration, and to facilitate its uptake into appropriate cells for the subsequent expression and presentation of antigen, in such a way as to elicit both systemic and mucosal antibody responses. These findings may have major implications for the design of novel vaccines and delivery strategies.

Oral delivery of poly(lactide-co-glycolide) encapsulated vaccines.

The induction of mucosal immune responses by oral delivery of vaccines is highly desirable. However vaccines to be used in this context will require protection from degradation in the gut and the use of specialised vehicles for their delivery and presentation. Using the biodegradable and biocompatible polymer, poly(lactide-co-glycolide), we have encapsulated bacterial and viral proteins, synthetic peptides and plasmid DNA in microparticles, and compared the immune responses resulting from their oral and parenteral administration to mice. The successful induction of specific systemic and mucosal humoral immune responses, as well as cell-mediated immune responses, demonstrates the potential of this polymer formulation as a vehicle for the oral delivery of vaccines.

Mucosal immunization with a measles virus CTL epitope encapsulated in biodegradable PLG microparticles.

The immunogenicity of a cytotoxic T cell epitope (CTL) representing residues 52-60 from measles virus (MV) nucleoprotein, encapsulated in poly(lactide-co-glycolide) (PLG) microparticles was evaluated after mucosal immunization. After intranasal administration of the encapsulated CTL epitope linked at the carboxyl terminus of two copies of a T-helper epitope (TT-NP6), peptide-specific and MV-specific CTL responses were detected in splenocytes. However, these responses were lower than the responses observed when the TT-NP6 peptide was administered intranasally in saline or using CTB as an adjuvant. Intranasal coadministration of the encapsulated TT-NP6 peptide with CTB did not result in any significant potentiation of the CTL responses. The effectiveness of biodegradable PLG microparticles for mucosal delivery of CTL epitopes, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic immunogens. However, further work is needed to define the requirements for effective absorption by the nasal epithelium.

Biodegradable microparticles as a delivery system for measles virus cytotoxic T cell epitopes.

Cytotoxic T-cell (CTL) responses are likely to be important for the clearance of a measles virus (MV) infection. To induce CTL responses. replicating vectors have generally been used but the use of such vectors in humans mav be problematic, and immunization with synthetic peptides may be more appropriate. We have investigated the potential of poly(lactide-co-glycolide)(PLG) microparticles as a delivery system for a CTL epitope representing residues 51-59 from MV nucleoprotein. After a single intraperitoneal injection in saline of the encapsulated epitope, CTL responses to the homologous peptide and MV were detected over a period of 4 months. Responses reached a maximum 30 days after priming and were maintained at high levels for 120 days. These responses were higher than those observed when the CTL epitope was administered in saline or as an emulsion in Incomplete Freund's Adjuvant. The pronounced immunostimulatory effect of microparticles, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic peptide immunogens.

Orally administered microencapsulated Bordetella pertussis fimbriae protect mice from B. pertussis respiratory infection.

Fimbriae from Bordetella pertussis have been encapsulated in poly(lactide-co-glycolide) microparticles of a size appropriate for uptake by the immune inductive tissues of the gastrointestinal tract. Mice were immunized by oral gavage with a single dose of 10 micrograms of microencapsulated fimbriae. The resulting immune responses were compared with those resulting from intraperitoneal injection of mice with equivalent amounts of fimbriae absorbed onto alhydrogel. The examination of serum and mucosal secretions, collected over a 6-week period, for specific antifimbrial antibodies clearly demonstrated that only orally immunized animals mounted measurable immune responses in external secretions. Six weeks after immunization, all immunized animals were protected against intranasal challenge with live B. pertussis.

Poly(lactide-co-glycolide) microencapsulation of vaccine antigens.

Fimbriae from Bordetella pertussis have been encapsulated in poly(lactide-co-glycolide) (PLG) microspheres of a size appropriate for oral administration. The binding of antibodies which react with conformational or linear fimbrial epitopes, to fimbriae released from microspheres, suggested that the process of was not detrimental to the native integrity of the protein. Mice were immunised by oral gavage with a single dose of microencapsulated fimbriae, or with fimbriae adsorbed onto alhydrogel and administered by intraperitoneal injection. The resulting immune responses in serum were comparable but only oral administration of microencapsulated fimbriae elicited specific immune responses in external secretions. Six weeks after immunisation, both groups of immunised animals were protected against challenge with live B. pertussis.

Human immunodeficiency virus infection of xenografted SCID-beige mice.

The application of C.B-17 SCID-Beige mice as an experimental animal system for the acceptance of human leukocyte xenografts, the establishment of functional human immune responses and infection with HIV has been assessed. Reconstitution efficiencies approaching 100% could be obtained by using 2 x 10(7) human peripheral blood lymphocytes (PBLs). Typical levels of human immunoglobulin in mouse blood reached 120 micrograms/ml within 2 weeks of reconstitution rising to a maximum in excess of 3 mg/ml by 5 weeks. Immunohistological examination of lung, spleen, lymph node and thymus tissue, derived from reconstituted mice, with human leukocyte specific monoclonal antibodies revealed the presence of human macrophages (CD68+), T cells (CD43+) and B cells (CD20+). The establishment of a functional immune system was demonstrated by the ability of reconstituted mice to respond to immunisation with KLH. Finally, reconstituted Hu-PBL-SCID-Beige mice were susceptible to infection with HIV-1 by intraperitoneal injection. These results indicate that SCID-Beige mice are a valuable tool for the generation of human/mouse chimeras and for the establishment of an in vivo HIV infection model. The results are compared with other similar model systems and are discussed in the context of animal models of HIV vaccine studies.

Efficient purification and rigorous characterisation of a recombinant gp120 for HIV vaccine studies.

A recombinant HIV-1 gp120 (rgp120) was expressed in a permanent Chinese Hamster Ovary (CHO) cell line (L761h) that constitutively secretes the product of clone p4 derived from the env gene of HIV-1 isolate GB8. The rgp120 was isolated from cell culture supernatants by a simple, rapid, non-denaturing and efficient purification procedure based on a novel combination of lectin affinity and FPLC ion-exchange chromatography. The purity of the isolated glycoprotein was rigorously confirmed by SDS-PAGE, capillary electrophoresis, laser desorption mass spectrometry, total amino acid analysis and N-terminal amino acid sequencing. The retention of biological activity by the purified rgp120 was assessed by determining the dissociation constant of rgp120 binding to sCD4. After formulation of this highly purified and biologically active rgp120 with "conventional" adjuvants, including types already used in clinical trials of candidate gp120-based HIV vaccines, antibody responses in immunised rabbits were analysed using panels of overlapping synthetic peptides. The consequences of using currently available adjuvants to deliver highly specialised and perhaps conformation-dependent molecules, like HIV gp120, are presented and discussed in the context of HIV vaccine development.

Experimental Legionnaires' disease in SCID-Beige mice reconstituted with human leucocytes.

A new small animal model of experimental Legionnaires' disease is described in which the reconstitution of SCID-Beige mice with human peripheral blood leucocytes permits the in-vivo growth of Legionella pneumophila in the lungs of aerosol-challenged mice. Following infection, viable bacterial counts within the lungs of mice increased from 10(5) cfu/lung at the time of inoculation to a maximum of 10(10) cfu/lung by 48 h post-inoculation. Two types of disease were detected in the lungs of infected SCID-Beige mice. An acute exudative bronchiolitis and bronchopneumonia were seen in the most severely affected mice and, in the less severely affected mice, lesions of subacute or chronic disease were seen with thickening of alveolar walls and consolidation of lung tissue. Human cells did not appear to be involved directly in the pathology but were required for the establishment of infection. Immunohistological staining of lung tissue revealed substantial amounts of bacterial antigen distributed in a pattern similar to that seen in human Legionnaires' disease.

Protection of mice from Bordetella pertussis respiratory infection using microencapsulated pertussis fimbriae.

Conditions have been established which allow the efficient entrapment of Bordetella pertussis fimbriae in poly(lactide-co-glycolide) microspheres. Fimbriae released from the matrix were found to have retained some degree of conformational structure, as determined by assessing the capacity of fimbrial protein to bind to antibodies mapping to either conformational or denatured structures on the fimbriae, either encapsulated in microspheres with a mean diameter of 24 microns and an estimated in vitro protein release rate of approximately 42 days, or conventionally adjuvanted with alhydrogel, elicited vigorous immune responses in mice. The encapsulated fimbriae appear to elicit marginally lower serum antibody levels than those induced by equivalent amounts of alhydrogel-adjuvanted fimbriae. Mice immunised with both preparations were, however, protected against intranasal infection with live B. pertussis as evidenced by the significant reduction in levels of bacterial colonisation observed in the lungs and tracheas of immunised animals when compared to the immunologically naive controls.

A molecular and serologic study of the envelope gene of the British isolate: HIV-1 GB8.

The entire envelope gene of a British HIV-1 isolate, GB8, was cloned, sequenced and aligned with those of the reference strains MN, SF2 and IIIB/LAI. Three of the viruses (MN, IIIB/LAI, GB8) and their recombinant gp120s, were then characterised using an extensive panel of human HIV-1 positive sera and mapped neutralising monoclonal antibodies (MAbs). Overall, the GB8 env-gene translation product shares 84% homology with those of the reference strains. Across the V3 region homology was greater between GB8 and SF2/MN (74.3-88.6%) than IIIB/LAI (63.9-66.7%). Accordingly, GB8 was sensitive to V3-specific MAbs which neutralise MN/SF2 and resistant to those that neutralize IIIB/LAI. In the CD4 binding region the central MWQEVGKAMYAPPI was conserved in all viruses but homology in the N-terminus was greater between GB8 and SF2 and IIIB/LAI than MN. GB8 and IIIB/LAI were sensitive to all MAbs raised against the CD4 binding site whereas MN was resistant to 3 of 4 tested. Human sera obtained from a London-based cohort did not differentiate between GB8 and MN in neutralisation assays, whereas IIIB/LAI titres were significantly lower at all stages of disease. These results show that GB8 carries a consensus-like V3 loop and is as representative as MN of HIV-1 viruses circulating in the UK. To our knowledge, GB8 is the only British HIV-1 isolate which has been characterised to date.

Purification and characterization of simian immunodeficiency virus (SIVmac) envelope glycoprotein gp130 from virus-infected cells.

A non-denaturing method has been developed for the purification of the envelope glycoprotein gp130 of the simian immunodeficiency virus (SIV) using infected cells as starting material. The procedure involves solubilization of cells infected with SIV (SIVmac251), enrichment of glycoproteins by lectin affinity chromatography, fractionation by reverse phase chromatography and purification by immunoaffinity chromatography. This procedure results in a greater than 95% purification of gp130 as assessed by polyacrylamide gel electrophoresis. There is no evidence for the presence of other virus-derived proteins after Western blot analysis using antibodies specific for virus proteins. Lectin-binding studies suggest that carbohydrate groups on the infected-cell-derived gp130 may differ from those on recombinant counterparts expressed in Chinese hamster ovary cells and Baculovirus-infected insect cells. The purified gp130 is highly immunogenic in rabbits and maintains the capacity to bind the CD4 receptor. A sufficient quantity of the infected-cell-derived gp130 has been prepared for immunization studies and subsequent live virus challenge studies in macaques.

The assembly of HIV within the Golgi apparatus and Golgi-derived vesicles of JM cell syncytia.

Chronic infection of the T-lymphocyte cell line JM with HIV-1 isolate GB8 results in the formation of multinucleate cells (syncytia). Transmission electron microscopy of these syncytia showed the presence of HIV particles both at the cell surface and within cytoplasmic vesicles. HIV particles were observed in dilated Golgi cisternae and Golgi-derived vesicles and in large vacuoles near the periphery of the syncytia. Immunolabelling was performed using an affinity-purified antiserum to the Golgi enzyme galactosyltransferase. This enzyme was consistently localized within both the Golgi apparatus and within virus-containing vesicles of JM syncytia, indicating that these vesicles originated from the Golgi apparatus.

Characterisation of a series of human immunodeficiency virus isolates derived sequentially from a single patient.

Five HIV-1 isolates were obtained sequentially from a single seropositive individual during the later stages of AIDS. Four of these isolates were adapted to grow in a continuous human T-lymphocytic cell line. Comparative biological and biochemical studies of the virus isolates were made using persistently infected cultures or virus derived from these systems respectively. The data obtained clearly shows that viruses with different biological properties can be isolated from the same individual at different times during the course of clinical AIDS.