Proteins synthesized and secreted during rat pancreatic development.

The synthesis and secretion of proteins during development of the pancreas was analyzed using two-dimensional gel electrophoresis. The pattern of synthesis of the total proteins of the pancreas was found to change very little from 14 to 18 d gestation. In addition, the protein synthetic pattern of the embryonic pancreas was very similar to the protein patterns of several other embryonic tissues (gut, lung, and mesenchyme). Between 18 d gestation and the adult stage, the synthesis of the majority of protein species fades as the synthesis of the secretory (pro)enzymes becomes dominant. Thus, the terminal differentiation of the pancreas appears to involve the dominant expression of a limited set of genes (coding, in part, for the digestive [pro]enzymes) while the pattern of expression of the remaining domain remains relatively unchanged. Many of the secretory (pro)enzymes were identified and their synthesis during development was monitored. The synthesis of several secretory proteins was detected between 15 and 18 d gestation (e.g., amylase and chymotrypsinogen), whereas the synthesis of others was not detected until after 18 d gestation (i.e., trypsinogen, ribonuclease, proelastase, and lipase). Between 18 d gestation and the adult stage, the synthesis of the digestive (pro)enzymes increases to > 90% of pancreatic protein synthesis. The secretion of digestive (pro)enzymes was detected as early as 15 d gestation. The selective release of a second set of proteins was detected in the early embryo. These proteins are not detected in the adult pancreas or in zymogen granules but are also released by several other embryonic tissues. The function of this set of proteins is unknown.

CpG-C ISS-ODN activation of blood-derived B cells from healthy and chronic immunodeficiency virus-infected macaques.

Cytosine-phosphate-guanine class C (CpG-C) immunostimulatory sequence oligodeoxynucleotides (ISS-ODNs) activate human B cells and dendritic cells (DCs), properties that suggest potential use as a novel adjuvant to enhance vaccine efficacy. After demonstrating that the CpG-C ISS-ODN C274 activates macaque DCs, we examined in vitro activation of macaque B cells by C274 as a prelude to evaluation of this molecule as an adjuvant in the testing of candidate human immunodeficiency virus vaccines in the rhesus macaque-simian immunodeficiency virus (SIV) model. C274 induced macaque CD20(+) B cells to proliferate more strongly than CD40 ligand or CpG-B ISS-ODN. C274 enhanced B cell survival; increased viability was most evident after 3-7 days of culture. Increased expression of CD40, CD80, and CD86 by B cells was apparent within 24 h of exposure to C274 and persisted for up to 1 week. C274-stimulated, B cell-enriched and peripheral blood mononuclear cell suspensions from naïve and immunodeficiency virus-infected monkeys secreted several cytokines [e.g., interleukin (IL)-3, IL-6, IL-12, interferon-alpha] and chemokines [e.g., monocyte chemoattractant protein-1/CC chemokine ligand 2 (CCL2), macrophage-inflammatory protein-1alpha/CCL3, IL-8/CXC chemokine ligand 8]. In comparison, exposure of macaque B cells to SIV had minimal impact on surface phenotype, despite inducing cytokine and chemokine production in cells from infected and uninfected animals. These observations emphasize the need to identify strategies to optimally boost immune function, as immunodeficiency viruses themselves only partially activate B cells and DCs. The ability of C274 to stimulate B cells and DCs in healthy and infected monkeys suggests its possible use as a broad-acting adjuvant to be applied in the rhesus macaque model for the development of preventative and therapeutic vaccines.

A comparison of glycosyltransferase activities and malignant properties in normal and transformed cells derived from BALB/c mice.

The ability of suspensions of BALB/c cells to catalyze the incorporation of nucleotide sugars into complex polysaccharides has been compared. These cells have previously been characterized for concanavalin A-induced agglutinability, tumorigenicity, and malignancy. All of the cell lines tested catalyze transfer of the sugar moieties of cytosine 5-monophosphate N-acetylneuraminic acid, uridine 5-diphosphate galactose, uridine 5-diphosphate N-acetylgalactosamine, uridine 5-diphosphate N-acetylglucosamine, uridine 5-diphosphate glucose, and guanidine 5-diphosphate monnose into glycoproteins and glycolipids. While some transformed lines exhibit alterations in transferase levels, others cannot be distinguished from normal cells. Normal cells, transformed cells that cause tumors that regress, and transformed cells that cause tumors that kill an immunologically competent host show growth-dependent changes in transferase activities. Determining the ability to catalyze carbohydrate transfer is insufficient for predicting the tumorigenic and malignant properties of a cell line.

Immunogenicity and protective efficacy of a recombinant filamentous haemagglutinin from Bordetella pertussis.

Bordetella pertussis is the causative agent of whooping cough, a major childhood pathogen; acellular vaccines consisting of purified B. pertussis antigens such as filamentous haemagglutinin (FHA) are commonly used to prevent pertussis. Despite the importance of FHA in B. pertussis pathogenesis and its inclusion in most acellular vaccines, the functional importance of individual domains in the induction of protective immunity is largely unknown. In this study, we have purified a recombinant FHA protein from Escherichia coli consisting of a 42 kDa maltose binding domain of E. coli and the 43 kDa type I immunodominant domain of FHA. The fusion protein (Mal85) was purified from E. coli cell lysates via affinity chromatography with an amylose column. Mal85 was then delivered to BALB/c mice intranasally encapsulated in liposomes, formulated with Protollin(TM) or in conjunction with an immunostimulatory CpG oligonucleotide. Mice were also vaccinated intraperitoneally with alum-adsorbed Mal85. Sera from all treatment groups showed strong IgG responses to Mal85 and recognized native FHA. Specific salivary IgA was induced in mice vaccinated with Mal85 in liposomes, Protollin(TM) and delivered with CpG. Vaccination with Mal85 encapsulated in liposomes or formulated with Protollin(TM) provided protection against aerosol challenge with B. pertussis in BALB/c mice. These data indicate that the type I domain of FHA is a protective antigen in mice and may serve as a candidate for inclusion in new acellular pertussis vaccines.

The adjuvant MF59 increases the immunogenicity and protective efficacy of subunit influenza vaccine in mice.

The immunogenicity and protective efficacy of influenza vaccine with and without the adjuvant MF59 was determined in mice. The addition of MF59 significantly increased the antibody response to the vaccine antigens over a wide dose range. Equivalent antibody titres were seen using 50- to 200-fold lower antigen concentrations when combined with MF59 compared with vaccine alone. The humoral response was sustained for at least 6 months after immunization. The addition of MF59 increased the protective efficacy of the vaccine: the amount of live virus detectable in the lungs of mice challenged with virus 1-6 months after immunization was reduced and the rate of survival was significantly increased. Influenza vaccine combined with MF59 gave full protection from viral challenge at antigen doses 65- to 80-fold lower than with vaccine alone.

Effects of dexamethasone and 5-bromodeoxyuridine on protein synthesis and secretion during in vitro pancreatic development.

Protein synthesis and secretion during in vitro pancreatic development and after treatment with the glucocorticoid dexamethasone and the thymidine analog 5-bromodeoxyuridine (BrdU) was monitored using two-dimensional gel electrophoresis. At 14 days gestation, the synthesis of more than 200 proteins and the secretion of a complex set of proteins was detected. The relative rate of synthesis and secretion of the majority of this set of proteins decreased dramatically during development; after 6 days of culture most were no longer detected. In contrast, the synthesis and secretion of pancreas-specific exocrine proteins amylase, a Sepharose binding protein (protein 2), and chymotrypsinogen first detected after one day in culture, increased throughout the 6-day culture period. Other pancreatic digestive (pro)enzymes normally found in the adult such as the basic form of chymotrypsinogen, lipase, ribonuclease, and trypsinogen were not detected during the culture period. Thus at least two distinct regulatory events are involved in the expression of the exocrine genes during development. Dexamethasone treatment during the 6-day culture period selectively increased the synthesis of amylase and several other minor secretory proteins. BrdU treatment caused major changes in the protein synthetic and secretory patterns of the pancreas as well as in morphogenesis. BrdU treated pancreases showed greatly reduced synthesis of amylase, protein 2, and chymotrypsinogen and prolonged synthesis of many proteins normally detected only at early stages of pancreatic development. BrdU treatment also stimulated the secretion of a set of proteins ostensibly associated with duct cells. Thus, BrdU specifically alters the developmental program of the pancreas.

Enhancement of humoral response against human influenza vaccine with the simple submicron oil/water emulsion adjuvant MF59.

Human influenza subunit vaccines are not fully protective in either the very young or elderly populations where risk is greatest. The use of an adjuvant to enhance antibody titer is an attractive option to increase vaccine efficacy. A series of squalene/H2O emulsions stabilized either by the amphipathic muramyl peptide MTP-PE (sodium N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanyl-2-(1',2'-dipalmitoyl- sn- glycero-3'phospho) ethylamide) or by mixtures of the sorbitan oleate surfactants Tween 80 and Span 85 have been tested as adjuvants with influenza vaccine. Combination of influenza vaccine with the Tween/Span stabilized emulsions has resulted in significantly higher antibody titers to vaccine in an extensive series of naive animal models. The use of submicron emulsion droplets is significant in determination of adjuvant activity while the presence of the muramyl peptide is not required for adjuvant activity. The 200-300 nm diameter emulsion formulation MF59 containing only the low toxicity components squalene, Tween 80 and Span 85 has been shown to enhance titers from 5 to 250 times that achievable with vaccine alone.

MF59 adjuvant enhances the immunogenicity of influenza vaccine in both young and old mice.

The responses of young (8 week) and old (18 month) mice to influenza vaccine with and without the potent emulsion adjuvant MF59 were compared. In influenza naive mice, vaccine-specific antibody and T-cell proliferation were significantly lower in the old group compared to the young group. Post-immunization cytokine levels and antibody isotype profiles were different in the old compared to the young mice. The addition of the adjuvant MF59, a submicron oil-in-water emulsion composed of 5% v/v squalene, 0.5% v/v Tween 80 and 0.5% v/v Span 85, significantly increased the immune responses of both the young and old naive mice to the vaccine. The responses of the old mice given adjuvant increased to levels equivalent to those of young mice with vaccine alone. In mice previously infected with influenza virus, similarly depressed immune responses to vaccination were detected in the old mice. While the addition of MF59 to the vaccine had little effect on antibody titres of the previously infected young mice, the adjuvant significantly increased the antibody responses of the previously infected old mice. These results suggest that influenza vaccine combined with MF59 may significantly improve immune responses of elderly humans to influenza vaccination.

MF59 adjuvant enhances the antibody response to recombinant hepatitis B surface antigen vaccine in primates.

The effect of the proprietary adjuvant MF59 on the immunogenicity of a recombinant hepatitis B virus (HBV) vaccine, PreS2+SAg, was investigated in baboons. The magnitude and duration of the antibody response to hepatitis B surface antigen (anti-HBs) induced by the HBV/MF59 vaccine was compared with the same antigen combined with alum and with two licensed alum vaccines. After one immunization, the HBV/MF59 vaccine generated anti-HBs titers >10 mIU/mL in a greater proportion of animals, and mean titers were 26- to 84-fold higher than titers from alum vaccines. After a third immunization, the HBV/MF59 vaccine generated titers 38- to 127-fold higher than alum vaccines. Seven months after the third immunization, HBV/MF59 elicited titers 75- to 472-fold higher than alum vaccines. The dramatic immune response elicited by HBV/MF59 in baboons suggests that MF59 may be a desirable adjuvant for use in improved HBV vaccines for humans.

A comparison of membrane components of normal and transformed BALB/c cells.

Membrane glycolipids, glycoproteins, and surface proteins of normal and transformed BALB/c cell lines have been compared. Several virally and spontaneously transformed cell lines showed differences in membrane components compared to normal A31 cells. These differences consisted of increased amounts of simpler gangliosides, absence of the large external transformation sensitive (LETS) protein, and the appearance of a major new glycoprotein band of about 105 000 molecular weight. In contrast, the spontaneously transformed cell line that caused the fastest growing tumors in vivo and the most rapid animal death (3T12T) did not have these changes. A31 and 3T12T glycolipid profiles appear similar as did glycoproteins and cell surface proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When Pronase-generated glycopeptides were analyzed by Sephadex G-50 chromatography, and enrichment in faster-eluting species was seen in two killing tumor lines (c5T and 3T12T) compared to A31. Regressing tumor lines (MSC, c5) did not show this change. Isolated membrane glycoproteins yield glycopeptides of different sized after Pronase digestion. In addition, several 3T12T glycoproteins yield glycopeptides that are larger than those from the corresponding glycoproteins of A31 cells. It appears that glycopeptide alterations associated with transformation occur in several membrane glycoproteins.

Pilot evaluation of influenza virus vaccine (IVV) combined with adjuvant.

The safety of licensed influenza virus vaccine (IVV) combined with a novel adjuvant containing muramyl tripeptide (MTP) conjugated to phosphatidylethanolamine (PE) was evaluated in a randomized pilot study. Ten healthy 23-30-year-old men were given a single intramuscular dose of IVV combined with saline (n = 5) or with 100 micrograms of MTP-PE in the MF59 adjuvant emulsion (MF59-100) (n = 5). Evaluations were performed on days 0, 1, 2, 4, 7 and 28 after inoculation. IVV alone was well tolerated. All volunteers immunized with IVV/MF59-100 experienced moderate to severe local and systemic reactions which interfered with usual activities. Discomfort at the injection site was first noted at 2-6 h; induration (5/5), erythema (3/5), and regional adenopathy (3/5) persisted for up to 4 days. Systemic symptoms including chills (5/5), fever (3/5), nausea (3/5) and/or dizziness (2/5) developed within 12 h of inoculation and resolved by 48 h. Elevated white blood cell count (days 1 and 2), erythrocyte sedimentation rate and serum fibrinogen were transiently observed. Although peak serum neutralizing antibody titres versus influenza A/H3N2 and influenza B antigens were higher in the group given IVV with MF59-100, these unexpected reactions indicate that this dose of adjuvant is unsuitable for use in combination with this IVV.

Systemic cytokine profiles in BALB/c mice immunized with trivalent influenza vaccine containing MF59 oil emulsion and other advanced adjuvants.

We have studied serum cytokine profiles in BALB/c mice after immunization with influenza vaccine alone or combined with the following adjuvants: alum; MF59 emulsion; MF59 containing the muramyl peptide N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2- dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy)) ethylamide (MTP-PE); MF59 plus the lipid A analogue monophosphoryl lipid A; MF59 plus the Quil A saponin fraction LTC; or LTC alone. Pooled mouse sera were analyzed by ELISA at various times after immunization for IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, and TNF-alpha. In naive mice, vaccine alone induced low levels of IL-3 and IL-5 only; vaccine plus alum induced a low IL-6 response as well. The MF59-based adjuvants significantly increased the IL-5 and IL-6 levels, whereas Quil A LTC induced strong IFN-gamma and measurable IL-2 responses, in addition to moderate IL-5 and IL-6. In previously infected mice, MF59 and MF59/MTP-PE were capable of generating IFN-gamma responses, as well as IL-5 and IL-6. All of the cytokine responses were rapid (peaking 3 to 12 h postimmunization) and short lived. In naive mice, the MF59 adjuvants induced serum cytokine profiles that are consistent with a primarily Th2-type response, whereas the Quil A LTC induced cytokines associated with both Th1 and Th2 responses. Ab analyses indicated that, although the adjuvants strongly affected the magnitude of the humoral response, there was no obvious correlation between the cytokine profile observed and the subclasses of Ab induced.

The influence of adjuvant on the therapeutic efficacy of a recombinant genital herpes vaccine.

The potential use of vaccines for treatment of chronic or persistent virus infections is an area of great interest and controversy. Previous experiments have shown that the incidence and severity of spontaneous recurrent genital herpes in latently infected guinea pigs could be significantly reduced by vaccination with herpes simplex virus glycoprotein subunit vaccines. The current study shows the critical role of adjuvant in an effective formulation. Immunization of previously infected guinea pigs with a subunit vaccine containing a muramyl peptide derivative, MTP-PE, in a low-oil emulsion as adjuvant reduced the incidence of recurrent disease up to 80% compared with formulations lacking MPT-PE. Vaccines containing adjuvant alone failed to modify recurrences. Alum, the traditional adjuvant, was not effective. Glycoprotein subunit vaccines elicited high-titer ELISA and neutralizing antibody responses far greater than those generated by virus infection. However, neither antibody titers nor lymphoproliferative responses reproducibly correlated with the pattern of recurrent disease.

Recombinant DNA approaches to feline leukemia virus immunization.

We have utilized 2 recombinant DNA strategies for immunization against FeLV in cats: (a) modified live virus was attenuated by mutation and recombination, and (b) an immunogen, consisting of subunit envelope protein, was prepared in genetically engineered yeast. Results indicated that the genetically manipulated live virus preparations were not protective against FeLV challenge because they were either not attenuated in virulence or were not sufficiently antigenic. Immunization with yeast-synthesized FeLV envelope protein followed by the modified live virus gave protective immunity in cats under experimental conditions. Future immunization attempts will concentrate on enhancing the immunogenic potency of the yeast- synthesized FeLV envelope protein.

Recent advances in vaccine adjuvants: the development of MF59 emulsion and polymeric microparticles.

Vaccines produced by recombinant DNA technology are safer than 'traditional' vaccines but they are often poorly immunogenic, requiring adjuvants to enhance their immunogenicity. Particulate adjuvants of defined dimensions (< 5 microns) have been shown to be effective in enhancing the immunogenicity of 'weak' antigens in animal models. Two novel adjuvants that possess significant potential for the development of new vaccines are the MF59 sub-microemulsion and polymeric microparticles. MF59 is an oil-in-water emulsion and has been shown to be both potent and safe in human subjects with several vaccines. Microparticles prepared from the biodegradable polymer poly(lactide-co-glycolide) have been shown to enhance immunogenicity when administered by mucosal routes, such as oral and intranasal, and they also possess considerable potential for the development of single-dose vaccines through the use of controlled-release technology.

Isolation and characterization of human papillomavirus type 6-specific T cells infiltrating genital warts.

The potential role of T cells in the control of human papillomavirus type 6 (HPV-6) infections is an appealing premise, but their actual role has been sparsely investigated. Since HPV-6 infections are confined to the epithelium, such an investigation should focus on the T cells present at the site of infection (i.e., the warts). Therefore, we isolated wart-infiltrating lymphocytes (WIL) from patients with clinically diagnosed anogenital warts. These WIL were characterized by their phenotype and their specificity for E7 and L1 proteins of HPV-6. The phenotype of WIL varied drastically from patient to patient, as determined by their expression of CD4, CD8, T-cell receptor alpha/beta chain (TCR alpha beta), and TCR gamma delta. Despite this heterogeneity in phenotype, HPV-6 E7 and/or L1-specific WIL, as determined by lymphoproliferation, could be isolated from more than 75% of the patients studied. Among all L1 peptides recognized by WIL, peptides 311-330 and 411-430 were the most consistently detected, with seven of nine patients for whom L1 peptide reactivity was observed responding to at least one of them. Moreover, the HPV-6 epitopic peptides recognized by WIL differed to some extent from those recognized by peripheral T cells.

Chemical and immunological studies of cell surfaces from normal and transformed cells.

Immunological and chemical studies of cell surfaces from normal and transformed BALB/c fibroblasts have shown alterations associated with transformation. The cells studied include normal lines which do not cause tumors when injected into BALB/c mice, viral transformants, and spontaneous transformants which cause tumors that either regress or grow progressively, killing the host. The spontaneously transformed progressors include cell lines which are immunogenic and nonimmunogenic as determined by the ability of tumor excision to protect an animal from subsequent rechallenge by tumor cells. Tumor-bearing mice produce lymphocytes which are nonspecifically cytotoxic for all the normal and transformed lines. Some of the cell lines induce specific antibody formation in BALB/hosts. Antisera have been prepared in rabbits which are specific for the transformed cell lines. These antisera can be used to determine specific surface changes on the transformed cells. Chemical studies have shown glycolipid alterations between the normal cells and some, but not all, of the transformants. Glycoproteins labeled by lactoperoxidase-125I or [3H] glucosamine were compared by SDS gel electrophoresis. Results from these studies do not show changes associated with malignancy. Individual glycoprotein regions from gels were treated with pronase, and the glycopeptides compared by Sephadex G50 chromatography. Alterations in glycopeptides from several cellular glycoproteins are the only changes which appear to be associated with malignancy.

The effect of adjuvants on the efficacy of a recombinant herpes simplex virus glycoprotein vaccine.

A recombinant, truncated HSV type 1 glycoprotein D secreted by Chinese hamster ovary cells (rgD1) was used to compare the ability of several adjuvants to stimulate protective immunity in guinea pigs. Adjuvants tested included CFA, aluminum hydroxide (alum), a lipophilic derivative of muramyl tripeptide (MTP-PE), and a muramyl dipeptide (MDP) covalently conjugated to rgD1. Animals were immunized three times with rgD1 plus the various adjuvants and antibody titers were determined by ELISA. Four weeks after the last immunization, the animals were challenged intravaginally with HSV type 2 and were monitored daily for clinical signs of disease, including frequency and severity of herpetic lesions, incidence of urinary retention, and mortality during the 14-day post-challenge observation period. Animals immunized in the foot-pad with rgD1 formulated with CFA showed the highest antibody titers. Animals immunized in the footpad with rgD1 using MTP-PE in a 4% squalene formulation, alum, or rgD1 conjugated to MDP showed mean antibody titers that were 57, 16, and 13% of the CFA titers, respectively. Immunization with rgD1 plus MTP-PE, alum, or rgD1-MDP conjugate by the i.m. route elicited lower antibody titers than the footpad route of immunization. Results of the viral challenge indicated that clinical symptoms of the groups immunized with rgD1 with CFA or MTP-PE as adjuvant were similar in magnitude and were markedly reduced compared with unimmunized control groups. Animals immunized with rgD1 combined with alum or rgD1-MDP conjugate showed clinical symptoms significantly more severe than the CFA or MTP-PE groups. The protective immunity observed after i.m. immunization of animals with rgD1 and MTP-PE was only slightly lower than animals immunized with the same Ag-adjuvant combination in the footpad. The results indicate that MTP-PE is an effective adjuvant for the recombinant herpes gD vaccine.

MF59 adjuvant enhances antibody responses of infant baboons immunized with Haemophilus influenzae type b and Neisseria meningitidis group C oligosaccharide-CRM197 conjugate vaccine.

The ability of the adjuvant MF59 to enhance the immunogenicity of polysaccharide-protein conjugate vaccines was investigated in infant baboons. MF59 consists of stable droplets (<250 nm) of the metabolizable oil squalene and two surfactants, polyoxyethylene sorbitan monooleate and sorbitan trioleate, in an oil-in-water emulsion. In humans, MF59 is well tolerated and enhances the immunogenicity of recombinant protein subunit or particle vaccines. Its effect on the immunogenicity of polysaccharide-protein conjugate vaccines is unknown. Baboons 1 to 4 months of age were immunized intramuscularly with Neisseria meningitidis group C and Haemophilus influenzae type b (Hib) oligosaccharide-CRM197 conjugate vaccines. The lyophilized vaccines were reconstituted with phosphate-buffered saline (PBS), Al(OH)3 (alum), or MF59. Groups of five animals each were given three injections of the respective formulations, with one injection every 4 weeks. Four weeks after each immunization, the MF59 group had up to 7-fold-higher geometric mean anticapsular-antibody titers than the alum group and 5- to 10-fold-higher N. meningitidis group C bactericidal-antibody titers. Twenty-one weeks after the third immunization, the MF59 group still showed 5- to 10-fold-higher anticapsular-antibody titers. The antibody responses of the animals given the vaccines reconstituted with PBS were low at all times measured. Both the MF59 and alum groups, but not the PBS group, showed booster antibody responses to unconjugated Hib and N. meningitidis group C polysaccharides, results consistent with induction of memory B cells. Thus, MF59 may be useful for accelerating and augmenting immunity to polysaccharide-protein conjugate vaccines in infants.