High Serum IgG Antibody Titers Research Articles

Liposome-based dry powder vaccine immunization targeting the lungs induces broad protection against pneumococcus

Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge.

Enhanced and long term immunogenicity of a Her-2/neu multi-epitope vaccine conjugated to the carrier CRM197 in conjunction with the adjuvant Montanide

BackgroundWe previously identified three short single peptides (P4, P6 and P7) representing different B-cell epitopes on the extracellular domain of Her-2/neu for a vaccine that was tested in a phase-I clinical trial. Here we describe the improvement of the multi peptide vaccine by fusing the single peptides to a hybrid peptide P467.MethodsAfter coupling to either virosomes or to diphtheria toxoid CRM197 (CRM), the hybrid peptide was tested in different concentrations in combination with either Montanide or Aluminium hydroxide (Alum) in preclinical studies.ResultsAlready low amount (10 μg) of P467 conjugated to CRM led to faster onset of high antibody levels compared to the P467-virosome. The formulation P467-CRM-Montanide induced higher serum IgG antibody titers, compared with P467-CRM-Alum, as examined by ELISA using recombinant Her-2/neu or Her-2/neu natively expressed on the tumor cell line SK-BR-3. Compared to P467-CRM-Alum, higher in vitro production of IL-2 and IFNγ in the Montanide-immunized mice was induced after re-stimulation of splenocytes with CRM but also with P467, indicating a clear Th1-biased response. In contrast to the single B cell peptides, the hybrid peptide led to T cell proliferation and cytokine production as CD4 T cell epitopes were generated in the fusion region of the single peptides P4 and P6 or P6 and P7. Additionally, a significantly higher proportion IFNγ-producing CD8+ T cells was found in the P467-CRM-Montanide immunized mice, probably by Montanide-driven bystander activation. Importantly, anti-P467 IgG antibodies exhibited anti-tumor properties and the combination of anti-P467 specific IgG with Herceptin® was found to inhibit the proliferation of Her-2/neu-overexpressing cell line SK-BR-3 in a significantly higher capacity than Herceptin® alone.ConclusionsFusion of the B cell peptides has led to additional generation of CD4 T cell epitopes, and this P467-multi epitope vaccine was found to induce polyclonal antibody responses with anti-proliferative capacity against Her-2/neu. The hybrid vaccine together with Montanide induced higher and long-lasting antibody levels, Th1-biased cellular responses being superior to vaccination with the single B cell peptides. This vaccine formulation is now planned to be evaluated in a phase Ib/II study in Her-2/neu overexpressing cancer patients.

Purification, Stability, and Immunogenicity Analyses of Five Bluetongue Virus Proteins for Use in Development of a Subunit Vaccine That Allows Differentiation of Infected from Vaccinated Animals

Bluetongue virus (BTV) causes bluetongue disease, a vector-borne disease of ruminants. The recent northerly spread of BTV serotype 8 in Europe resulted in outbreaks characterized by clinical signs in cattle, including unusual teratogenic effects. Vaccination has been shown to be crucial for controlling the spread of vector-borne diseases such as BTV. With the aim of developing a novel subunit vaccine targeting BTV-8 that allows differentiation of infected from vaccinated animals, five His-tagged recombinant proteins, VP2 and VP5 of BTV-8 and NS1, NS2, and NS3 of BTV-2, were expressed in baculovirus or Escherichia coli expression systems for further study. Optimized purification protocols were determined for VP2, NS1, NS2, and NS3, which remained stable for detection for at least 560 to 610 days of storage at +4°C or -80°C, and Western blotting using sera from vaccinated or experimentally infected cattle indicated that VP2 and NS2 were recognized by BTV-specific antibodies. To characterize murine immune responses to the four proteins, mice were subcutaneously immunized twice at a 4-week interval with one of three protein combinations plus immunostimulating complex ISCOM-Matrix adjuvant or with ISCOM-Matrix alone (n = 6 per group). Significantly higher serum IgG antibody titers specific for VP2 and NS2 were detected in immunized mice than were detected in controls. VP2, NS1, and NS2 but not NS3 induced specific lymphocyte proliferative responses upon restimulation of spleen cells from immunized mice. The data suggest that these recombinant purified proteins, VP2, NS1, and NS2, could be an important part of a novel vaccine design against BTV-8.

Stability Kinetics of Influenza Vaccine Coated onto Microneedles During Drying and Storage

This study sought to determine the effects of microneedle coating formulation, drying time and storage time on antigen stability and in vivo immunogenicity of influenza microneedle vaccines. The stability of inactivated influenza virus vaccine was monitored by hemagglutination (HA) activity and virus particle aggregation as a function of storage time and temperature with or without trehalose. In vivo immunogenicity of inactivated influenza vaccines coated onto microneedles was determined in mice by virus-specific antibody titers and survival rates. In the absence of trehalose, HA activity decreased below 10% and to almost zero after 1h and 1month of drying, respectively. Addition of trehalose maintained HA activity above 60% after drying and above 20% after 1month storage at 25°C. Loss of HA activity generally correlated with increased virus particle aggregation. Administration of microneedles coated with trehalose-stabilized influenza vaccine yielded high serum IgG antibody titers even after 1month storage, and all animals survived with minimal weight loss after lethal challenge infection. Inactivated influenza virus vaccine coated on microneedles with trehalose significantly improved the HA activity as well as in vivo immunogenicity of the vaccine after an extended time of storage.

Development of a Liposaccharide-Based Delivery System and Its Application to the Design of Group A Streptococcal Vaccines

Group A streptococcus (GAS) is associated with many human diseases, ranging in severity from benign to life-threatening. A promising strategy for developing vaccines against GAS involves the use of carbohydrates as carriers for peptide antigens. This study describes the optimized synthesis of d-glucose and d-galactose derived carriers, bearing an adipate linker and four tert-butoxycarbonyl protected aminopropyl groups. Prophylactic GAS vaccine candidates were synthesized by conjugating multiple copies of a single GAS M protein derived peptide antigen (either J8 or J14) onto the carbohydrate carriers. These antigens contain peptide sequences, which are highly conserved and offer the potential to prevent infections caused by up to 70% of GAS strains. Lipophilic amino acids were also conjugated to the d-glucose anomeric carbon to produce a self-adjuvanting liposaccharide vaccine. High serum IgG antibody titers against each of the incorporated peptide epitopes were detected following subcutaneous immunization of B10.BR (H-2 (k)) mice with the liposaccharide vaccine candidates.

Synthesis and in vivo studies of carbohydrate‐based vaccines against group A Streptococcus

Carbohydrates, as carriers, providing numerous attachment points for the conjugation of peptide antigens and their optimal orientation for the recognition by cells of the immune system, reducing degradation of the attached peptide antigens and many other advantages make carbohydrate-based vaccine highly promising approach. Multiple copies of a single group A streptococcal (GAS) M protein derived specific peptide antigens (J8 or J14) were coupled onto carbohydrate cores (D-glucose and D-galactose) linked to lipophilic amino acids to produce a self-adjuvanting liposaccharide vaccine against GAS strains. In vivo experiments showed high serum IgG antibody titers against each of the incorporated peptide epitopes, J8 or J14.

Synthesis of a Highly Pure Lipid Core Peptide Based Self-Adjuvanting Triepitopic Group A Streptococcal Vaccine, and Subsequent Immunological Evaluation

We have developed a highly pure, self-adjuvanting, triepitopic Group A Streptococcal vaccine based on the lipid core peptide system, a vaccine delivery system incorporating lipidic adjuvant, carrier, and peptide epitopes into a single molecular entity. Vaccine synthesis was performed using native chemical ligation. Due to the attachment of a highly lipophilic adjuvant, addition of 1% (w/v) sodium dodecyl sulfate was necessary to enhance peptide solubility in order to enable ligation. The vaccine was synthesized in three steps to yield a highly pure product (97.7% purity) with an excellent overall yield. Subcutaneous immunization of B10.BR (H-2(k)) mice with the synthesized vaccine, with or without the addition of complete Freund's adjuvant, elicited high serum IgG antibody titers against each of the incorporated peptide epitopes.

Method for the Synthesis of Multi-EpitopicStreptococcuspyogenesLipopeptide Vaccines Using Native Chemical Ligation

The aim of this study was to investigate methods for the synthesis of highly pure, well-characterized analogues of the lipid core peptide (LCP) system. Difficulties synthesizing and purifying conventional LCP systems have led to the requirement for a technique to produce highly pure, LCP-based vaccines for potential use in human clinical trials. The current study describes methods for the attachment of lipophilic adjuvants onto multi-epitopic peptide vaccines. Described is the synthesis, using native chemical ligation, of a highly pure, tri-epitopic, group A streptococcal (GAS) lipopeptide vaccine candidate. Intranasal immunization of the described tri-epitopic GAS lipopeptide with the mucosal adjuvant cholera toxin B subunit induced high serum IgG antibody titers specific for each of the incorporated peptide epitopes.

Immunogenicity and protective efficacy of a formalin-inactivated rotavirus vaccine combined with lipid adjuvants

The immunogenicity and protective efficacy of inactivated rotavirus vaccine administered intramuscularly with lipid adjuvants; MPL ® (monophosphoryl lipid A from Salmonella minnesota) or L3 ® (monooleate/lauric acid) was evaluated in an infant mouse model. Purified and formalin-inactivated rhesus rotavirus (I-RRV) combined with one of the adjuvants were administered to female balb/c mice at 0, 4 and 8 weeks. High serum IgG antibody titers developed in all vaccinated groups; I-RRV (GMT 45,524±9,819), I-RRV-MPL ® (GMT 190,637±64,250) and I-RRV-L3 ® (GMT 126,266±27,553). The formalin-inactivation procedure preserved neutralizing epitopes and elicited high neutralizing antibody titers; I-RRV (GMT 43,053 S.E.M.±4,189), I-RRV-MPL ® (GMT 66,398 S.E.M.±20,202) and I-RRV-L3 ® (GMT 60,887 S.E.M.±10,750). All offsprings to immunized dams were protected against clinical diarrhea upon oral challenge with RRV. The IgG1/IgG2a ratio was in all immunized groups ∼1 suggesting development of a balanced Th1/Th2 response.

Evidence ofPasteurella haemolyticalinked immune complex disease in natural and experimental models

The pathogenesis of bovine pneumonic pasteurellosis is not completely understood, and studies have not established thatPasteurella haemolyticaA1 (Ph1) virulence is exclusively responsible for the development of acute pulmonary lesions. The purpose of this investigation was to determine if immune complex disease is involved in the pathogenesis of bovine pneumonic pasteurellosis. A retrospective immunohistologic study of lung tissue from natural cases of bovine pneumonic pasteurellosis (44) as performed, and immune complexes were observed in alveloar spaces and walls in 88% of these cases. To study this pathologic mechanism experimentally, groups of mice were immunized with purified Ph1 outer membranes (OMs) or sham immunized on days 0 and 14. Mice were challenged intratracheally on day 24 with either live Ph1 or Ph1 OMs, and pulmonary lesions were assessed 24 h after challenge. Placebo immunized mice developed focal infiltrates of neutrophils and macrophages centered around large caliber bronchi. Mice immunized with Ph1 OMs and challenged with live Ph1 or OMs developed severe bronchointerstitial pneumonia with diffuse neutrophilic infiltration, focal necrosis, hemorrhage and edema, that is histologically similar to bovine pneumonic pasteurellosis. Immunohistology revealed flocculent aggregates of IgG and complement positive material within alveolar spaces and walls from mice challenged with live Ph1, and fine granular deposits of IgG and complement positive material were observed lining the alveolar walls from mice challenged with Ph1 OMs. Immunized mice exhibited high serum IgG antibody titers to Ph1 outer membrane proteins (OMPs). Results of this study suggest that immune complex disease plays a role in the pathogenesis of bovine pneumonic pasteurellosis.

Influence of inoculation route on the carrier state of Salmonella choleraesuis in swine

This study was designed to investigate the carrier state of swine infected with Salmonella choleraesuis. Thirty-five pigs were divided into 3 groups. Groups 1 ( n = 15) and 2 ( n = 16) were challenged with 10 8 CFU of S. choleraesuis intranasally or by gastric route, respectively. Group 3 ( n = 4) served as uninoculated controls. Pigs were necropsied at 2, 4, 6, and 12 weeks post inoculation. Clinical signs and microscopic lesions were more severe for group 1. Salmonella choleraesuis was recovered from a greater percentage of tissue samples for group 1 versus group 2 at 2, 4, and 6 weeks post inoculation. No differences were observed between groups at 12 weeks post inoculation. Regardless of route of inoculation, S. choleraesuis was most often recovered from the ileocolic junction, ileocolic lymph node, cecal contents, tonsil, lung and colon. Both groups shed S. choleraesuis in the feces sporadically throughout the 12 week period indicating that a carrier state is maintained for at least 12 weeks. However, group 1 shed higher numbers of S. choleraesuis initially. Serum IgG, IgM, and IgA antibody responses to S. choleraesuis lipopolysaccharide and heat extract antigens were observed for both groups. Higher serum IgG antibody titers to S. choleraesuis lipopolysaccharide were observed for group 2. Intestinal antibody responses for both groups included IgG and IgM responses but not an IgA response. Both routes of inoculation stimulated peripheral blood B-cells while the intranasal route (group 1) was more effective at stimulating peripheral blood T-cells. The reduction in levels of tissues infection and shedding observed for both groups coincided with the development of the host immune response. These data indicate that route of inoculation affects the development of humoral and cellular immunity, influences levels of Salmonella shed into the environment and the distribution of Salmonella within tissue.

Antibody in cerebrospinal fluid in non-meningitic coccidioidomycosis.

The cerebrospinal fluid (CSF) of 19 patients with non-meningeal coccidioidomycosis was found to contain coccidioidal antibodies. The complement fixation test was negative with these specimens but antibodies could be demonstrated after concentrating the CSF and testing by immunodiffusion. The serum complement fixation titer of these patients was generally 1:16 or higher. Five additional patients with non-meningeal coccidioidomycosis had no detectable coccidioidal antibody in their CSF by complement fixation or immunodiffusion. It is suggested that with sufficiently high serum IgG antibody titers, antibody may pass into the CSF in the absence of CNS involvement.