Maternal immunization with Group B Streptococcus six-valent polysaccharide conjugate vaccine supported by lack of toxicity in rat and rabbit fertility and developmental toxicity studies.

A maternal Group B Streptococcus (GBS) six-valent polysaccharide conjugate vaccine (GBS6) is being developed to protect neonates and infants up to 3 months of age through passive transfer of antibodies from the mother to the infant. Fertility and developmental toxicity studies were conducted in female Sprague Dawley rats and New Zealand White rabbits with GBS6 (20 µg capsular polysaccharide/serotype formulated with or without AlPO4 , the highest clinical dose). Females were administered the full human dose of the GBS6 formulation intramuscularly twice prior to mating and twice during gestation, to ensure that high antibody levels were maintained throughout gestation and lactation. Approximately, half of the rats and rabbits were evaluated at the end of gestation, and the remainder were evaluated at the end of lactation. Maternal blood for GBS6 serology, to measure antibody titers to the GBS6 antigens, was collected prior to the first dose, prior to mating, and at each necropsy. Blood for serology was also collected from offspring at the end of gestation and lactation. In both species, there was no evidence of vaccine-related effects on fertility, embryo-fetal development, or postnatal development of the offspring, supporting regulatory guidance that single-species evaluation would have been sufficient. Functional serum antibodies to all six serotypes in the vaccine were confirmed in maternal animals and functional serum antibodies to one or more of the six serotypes was also confirmed in some rat offspring and most of the rabbit offspring. The results of these studies supported the safety of GBS6 vaccine administration to pregnant women.

Conjugation, characterization and toxicity of lipophosphoglycan-polyacrylic acid conjugate for vaccination against leishmaniasis.

Research on the conjugates of synthetic polyelectrolytes with antigenic molecules, such as proteins, peptides, or carbohydrates, is an attractive area due to their highly immunogenic character in comparison to classical adjuvants. For example, polyacrylic acid (PAA) is a weak polyelectrolyte and has been used in several biomedical applications such as immunological studies, drug delivery, and enzyme immobilization. However, to our knowledge, there are no studies that document immune-stimulant properties of PAA in Leishmania infection. Therefore, we aimed to develop a potential vaccine candidate against leishmaniasis by covalently conjugating PAA with an immunologically vital molecule of lipophosphoglycan (LPG) found in Leishmania parasites. In the study, LPG and PAA were conjugated by a multi-step procedure, and final products were analyzed with GPC and MALDI-TOF MS techniques. In cytotoxicity experiments, LPG-PAA conjugates did not indicate toxic effects on L929 and J774 murine macrophage cells. We assume that LPG-PAA conjugate can be a potential vaccine candidate, and will be immunologically characterized in further studies to prove its potential.

A novel combined conjugate vaccine: enhanced immunogenicity of bFGF with CRM197 as a carrier protein.

Tumor growth is partly dependent on tumor-associated angiogenesis, which is regulated by angiogenic growth factors. As the first angiogenic growth factor to be identified, basic fibroblast growth factor (bFGF) plays a major role in angiogensis and tumor growth and has been an effective target for anti-tumor therapy. However, due to its low immunogenicity, injection with bFGF alone cannot stimulate the body to produce a strong immune response. In this study, we investigated the role of CF (containing bFGF and CRM197) assisted by CpG and alum in enhancing antigen-specific immune response and suppressing the growth of murine colon carcinoma. The results revealed that compared to bFGF, CF could not stimulate NIH-3T3 fibroblast proliferation even at a concentration of 10 µg/ml in vitro. In vivo, the CF-CpG-alum produced a stronger antigen-specific immune response and inhibited tumor growth. The anti-tumor activity was associated with generating antigen-specific antibody, suppressing angiogenesis, promoting the apoptosis of tumor cells and inducing the mixed Th1 and Th2 responses. This indicates that CRM197 may be an innovative intramolecular adjuvant and provides a rational preservation for mouse CT26 colon carcinoma.

Mannan-Abeta28 conjugate prevents Abeta-plaque deposition, but increases microhemorrhages in the brains of vaccinated Tg2576 (APPsw) mice.

BACKGROUND: New pre-clinical trials in AD mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse responses that occurred during the clinical trials with AN-1792 vaccine formulation. Recently, we have pursued an alternative immunization strategy that replaces QS21 the Th1 type adjuvant used in the AN-1792 clinical trial with a molecular adjuvant, mannan that can promote a Th2-polarized immune response through interactions with mannose-binding and CD35/CD21 receptors of the innate immune system. Previously we established that immunization of wild-type mice with mannan-Abeta28 conjugate promoted Th2-mediated humoral and cellular immune responses. In the current study, we tested the efficacy of this vaccine configuration in amyloid precursor protein (APP) transgenic mice (Tg2576). METHODS: Mannan was purified, activated and chemically conjugated to Abeta28 peptide. Humoral immune responses induced by the immunization of mice with mannan-Abeta28 conjugate were analyzed using a standard ELISA. Abeta42 and Abeta40 amyloid burden, cerebral amyloid angiopathy (CAA), astrocytosis, and microgliosis in the brain of immunized and control mice were detected using immunohistochemistry. Additionally, cored plaques and cerebral vascular microhemorrhages in the brains of vaccinated mice were detected by standard histochemistry. RESULTS: Immunizations with low doses of mannan-Abeta28 induced potent and long-lasting anti-Abeta humoral responses in Tg2576 mice. Even 11 months after the last injection, the immunized mice were still producing low levels of anti-Abeta antibodies, predominantly of the IgG1 isotype, indicative of a Th2 immune response. Vaccination with mannan-Abeta28 prevented Abeta plaque deposition, but unexpectedly increased the level of microhemorrhages in the brains of aged immunized mice compared to two groups of control animals of the same age either injected with molecular adjuvant fused with an irrelevant antigen, BSA (mannan-BSA) or non-immunized mice. Of note, mice immunized with mannan-Abeta28 showed a trend toward elevated levels of CAA in the neocortex and in the leptomeninges compared to that in mice of both control groups. CONCLUSION: Mannan conjugated to Abeta28 provided sufficient adjuvant activity to induce potent anti-Abeta antibodies in APP transgenic mice, which have been shown to be hyporesponsive to immunization with Abeta self-antigen. However, in old Tg2576 mice there were increased levels of cerebral microhemorrhages in mannan-Abeta28 immunized mice. This effect was likely unrelated to the anti-mannan antibodies induced by the immunoconjugate, because control mice immunized with mannan-BSA also induced antibodies specific to mannan, but did not have increased levels of cerebral microhemorrhages compared with non-immunized mice. Whether these anti-mannan antibodies increased the permeability of the blood brain barrier thus allowing elevated levels of anti-Abeta antibodies entry into cerebral perivascular or brain parenchymal spaces and contributed to the increased incidence of microhemorrhages remains to be investigated in the future studies.

Pilot study of a heptavalent vaccine-keyhole limpet hemocyanin conjugate plus QS21 in patients with epithelial ovarian, fallopian tube, or peritoneal cancer.

PURPOSE: To characterize the safety and immunogenicity of a heptavalent antigen-keyhole limpet hemocyanin (KLH) plus QS21 vaccine construct in patients with epithelial ovarian, fallopian tube, or peritoneal cancer in second or greater complete clinical remission. EXPERIMENTAL DESIGN: Eleven patients in this pilot trial received a heptavalent vaccine s.c. containing GM2 (10 microg), Globo-H (10 microg), Lewis Y (10 microg), Tn(c) (3 microg), STn(c) (3 microg), TF(c) (3 microg), and Tn-MUC1 (3 microg) individually conjugated to KLH and mixed with adjuvant QS21(100 microg). Vaccinations were administered at weeks 1, 2, 3, 7, and 15. Periodic blood and urine samples were obtained to monitor safety (complete blood count, comprehensive panel, amylase, thyroid-stimulating hormone, and urinalysis) and antibody production (ELISA, fluorescence-activated cell sorting, and complement-dependent cytotoxicity). RESULTS: Eleven patients were included in the safety analysis; 9 of 11 patients remained on study for at least 2 weeks past fourth vaccination and were included in the immunologic analysis (two withdrew, disease progression). The vaccine was well tolerated. Self-limited and mild fatigue (maximum grade 2 in two patients), fever, myalgia, and localized injection site reactions were most frequent. No clinically relevant hematologic abnormalities were noted. No clinical or laboratory evidence of autoimmunity was seen. Serologic responses by ELISA were largely IgM against each antigen with the exception of Tn-MUC1 where both IgM and IgG responses were induced. Antibody responses were generally undetectable before immunization. After immunization, median IgM titers were as follows: Tn-MUC1, 1:640 (IgG 1:80); Tn, 1:160; TF, 1:640; Globo-H, 1:40; and STn, 1:80. Only one response was seen against Lewis Y; two were against GM2. Eight of nine patients developed responses against at least three antigens. Antibody titers peaked at weeks 4 to 8 in all patients. Fluorescence-activated cell sorting and complement-dependent cytotoxicity analysis showed substantially increased reactivity against MCF7 cells in seven of nine patients, with some increase seen in all patients. CONCLUSIONS: This heptavalent-KLH conjugate plus QS21 vaccine safely induced antibody responses against five of seven antigens. Investigation in an adequately powered efficacy trial is warranted.

Phase I study of a lipooligosaccharide-based conjugate vaccine against nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) accounts for about one-third of purulent otitis media (OM) in children and is a common cause of pulmonary infection in adults with decreased resistance. Based upon sero-epidemiological data in humans and immunochemical data in laboratory animals, a lipooligosaccharide (LOS)-tetanus toxoid (TT) conjugate was prepared and evaluated for its safety and immunogenicity in a Phase I study of 40 healthy adults. The conjugate was injected intramuscularly into all volunteers: 28 of them received a second injection 14 weeks later. Local and systemic reactions were monitored and sera, taken before and 2, 6, 14, 16, and 38 weeks after injection, were assayed for IgG, IgA, and IgM antibodies to the LOS by ELISA and for bactericidal activity. The results indicate that there were no significant local or systemic reactions after either injection. All volunteers had pre-existing IgG anti-LOS. The geometric mean (GM) level rose from 14 to 40 at 2 weeks, remained at 35 at 6 weeks (40 or 35 versus 14, P<0.01) and dropped to 27 at 14 weeks after the first injection. There was also a rise 2 weeks after the second injection (27 versus 37, P<0.05). A total of 52.5% of subjects showed serum-conversion (greater than four-fold increase) after one and two injections. At 38 weeks, the GM IgG anti-LOS was still higher than before initial injection (20 versus 14, P<0.05). A similar pattern of reactivity was observed for IgA and IgM anti-LOS. Similar to that observed in mice, but not in rabbits, the conjugate-induced antibodies did not yield significant bactericidal activity in vitro. The LOS-TT conjugate is well tolerant in adults and a Phase II evaluation of the conjugate in children is planned.

Conjugates of aberrant gangliosides in antiglioma vaccine: toxicological assay.

We studied sterility and toxicity of vaccine LS1 containing aberrant gangliosides isolated from brain bioptates of 48 patients with gliomas of different malignancy and covalently bound to keyhole limpet hemocyanin. The vaccine was safe. This preparation produced no side effects in experimental animals. Our findings substantiated the necessity of father development of this method of vaccination. The vaccine should undergo clinical tests in patients with malignant gliomas.

A revisit of mucosal IgA immunity and oral tolerance.

Induction of mucosal immunity by oral immunization with protein antigen alone is difficult: potent mucosal adjuvants, vectors, or other special delivery systems are required. Cholera toxin (CT) has been shown to be an effective adjuvant for the development of mucosal vaccines and, when given with vaccine, induces both mucosal and systemic immune responses via a Th2 cell-dependent pathway. However, and in addition to potential type-I hypersensitivity, a major concern for use of mucosal adjuvants such as CT is that this molecule is not suitable for use in humans because of its inherent toxicity. When we examined the potential toxicity of CT for the central nervous system, both CT and CT-B accumulated in the olfactory nerves/epithelium and olfactory bulbs of mice when given by the nasal route. The development of effective mucosal vaccines for the elderly is also an important issue; however, only limited information is available. When mucosal adjuvanticity of CT was evaluated in aged mice, an early immune dysregulation was evident in the mucosal immune system. The present review discusses these potential problems for effective mucosal vaccine development. Tolerance represents the most common and important response of the host to environmental antigens, including food and commensal bacterial components, for the maintenance of an appropriate immunological homeostasis. We have examined whether Peyer patches could play a more important role for the maintenance of oral tolerance. Using Peyer patch-null mice, we found that mice lacking this gut-associated lymphoid tissue retained their capability to produce secretory IgA antibodies but did not develop normal oral tolerance to protein antigens.

IgA nephropathy in mice following repeated administration of conjugated Haemophilus influenzae type B vaccine (PRP-T).

OBJECTIVE: In this study, the risk of IgA nephropathy in Swiss albino mice following the subcutaneous administration of conjugated Haemophilus influenzae type b vaccine (PRP-T), containing capsular polysaccharide of the organism (PRP) conjugated to tetanus protein (T), was evaluated. METHODS: Three treatment and corresponding control groups, each containing mice, were constituted and given 2, 4, 6 injections of 1/4 HD of PRP-T or placebo, respectively, at 2-week intervals. All mice in each treatment group were sacrificed two weeks from the last injection to examine sequential glomerular changes. RESULTS: The niceoscpic examination of renal tissues revealed mesangial proliferation (6/7; 85%) in the first group given 2 doses of vaccine; mesangial proliferation (5/7; 72%) and increase in matrix (7/7; 100%) in the second group given 4 doses; and mesangial proliferation (7/7; 100%), increase in matrix (7/7; 100%), IgA (7/7; 100%) and C3 (3/7; 42%) deposition within mesangium in the third group given 6 doses. No histopathological changes were detected in the renal tissues of any control mouse. When the experimental groups were compared statistically with their respective controls at the light microscopic level, mesangial proliferation in the first group (p: 0.0047), mesangial proliferation (p: 0.021) and increase in matrix (p: 0.001) in the second group, mesengial proliferation (p: 0.001) and increase in matrix (p: 0.001) in the third group were determined to be significantly different. When study and control groups were compared by immunofluorescence microscopy, only the third group revealed a statistically significant difference with respect to IgA deposition (p: 0.001). C3 deposition was also demonstrated in this group, but it was not significantly different (p: 0.192). However, in no instance was a control mouse found to have any form of immune deposition. CONCLUSION: We concluded that conjugated Haemophilus influenzae type b vaccine, given at two-week intervals to a total of six doses, caused secondary IgA nephropathy in mice.