The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has bee... more The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb4...
Immunostimulating complexes were prepared with antigens extracted from tachyzoites of Toxoplasma ... more Immunostimulating complexes were prepared with antigens extracted from tachyzoites of Toxoplasma gondii and were used to immunize mice. The major antigens incorporated into the immunostimulating complexes were the P30 and P22 antigens and an antigen with an approximate molecular weight of 6,000. Other antigens of molecular weights above 30,000 were also present. High antibody titers to T. gondii antigens and a delayed-type hypersensitivity reaction were noted for the immunized mice. Challenge of these mice with tachyzoites injected interperitoneally or with oocysts administered orally resulted in a statistically significant (P < 0.001) conditional probability of survival compared with that of controls. In contrast, the differences between immunized mice and controls challenged with tissue cysts did not attain statistical significance.
B- and T-cell responses have been studied after primary and secondary immunizations of mice with ... more B- and T-cell responses have been studied after primary and secondary immunizations of mice with iscoms containing influenza virus envelope glycoproteins. After primary immunization both B- and T-cell responses were initiated in the draining lymph nodes. T-cells showed peak activity with respect to proliferation and cytokine production after five to eight days and the highest number of IgG secreting cells (IgG-SC) was recorded at day seven. The responses in the spleen developed slowly but were of longer duration. Cytokines produced by spleen cells included high levels of IFN-gamma and IL-2. After a secondary immunization the frequencies of IgG-SC were drastically increased in both LN and spleen, but decreased rapidly with time. At day eight after the secondary immunization high numbers of IgG-SC were detected in the bone marrow. High titres of IgG1 and IgG2a and substantial titres of IgG2b and IgG3 were detected in serum.
ABSTRACTBluetongue virus (BTV) causes bluetongue disease, a vector-borne disease of ruminants. Th... more ABSTRACTBluetongue 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 orEscherichia coliexpression 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 ...
Alignment of B-strains based on hemagglutinin amino acid sequence. B/Bris = B/Brisbane/60/08 [Gen... more Alignment of B-strains based on hemagglutinin amino acid sequence. B/Bris = B/Brisbane/60/08 [GenBank: ACN29380.1], B/Mal = B/Malaysia/2506/04 [GenBank: ACR15732], B/Florida = B/Florida/04/06 [GenBank: ACF54246.1], B/Mass = B/Massachusetts/02/12 [GenBank: AGL06036.1]. Blue bar indicates head domain defined as Asn58 to Glu307. (PDF 156 kb)
Body weight loss and clinical scores following homologous challenges. Mice (6 or 10/group) were i... more Body weight loss and clinical scores following homologous challenges. Mice (6 or 10/group) were immunized once with different doses of seasonal trivalent virosomal influenza vaccine (TVV) with or without Matrix-M™ or PBS and challenged with vaccine-homologous virus strains H1N1 A/Netherlands/602/09, H3N1 A/Perth/16/09 or B/Malaysia/2506/04 and monitored for 21 days for survival, body weight loss and clinical symptoms. Graphs represent mean body weight changes with 95 % confidence interval and median clinical scores with interquartile range. The HA amino acid sequences of H1N1 A/Netherlands/602/09 and H1N1 A/California/07/09 differ by 5 residues (99.5 % homology), those of H3N2 A/Perth/16/09 and H3N2 A/Victoria/210/09 differ by 7 residues (98.8 % homology), and those of B/Malaysia/2506/04 and B/Brisbane/60/08 differ by 5 residues (99.2 % homology) (Additional file 4: Figure S3, Additional file 5: Figure S4, Additional file 6: Figure S5). (PDF 96 kb)
Alignment of H3N2 strains based on hemagglutinin amino acid sequence. H3/Vic = A/Victoria/210/09 ... more Alignment of H3N2 strains based on hemagglutinin amino acid sequence. H3/Vic = A/Victoria/210/09 [GenBank: AFM72883.1], H3/Perth = A/Perth/16/09 [GenBank: ACS71642.1], H3/HK = A/Hong Kong/1/68 [GenBank: ACU79871.1]. Blue bar indicated head domain defined as Cys68 to Cys293. (PDF 146 kb)
Additional file 1. Gene expression in pooled blood samples from pigs administered Matrix-M or sal... more Additional file 1. Gene expression in pooled blood samples from pigs administered Matrix-M or saline. The gene expression was analysed by a qPCR plate array in pooled blood samples collected at 0, 18, 30 h, 2 and 3 days after administration of Matrix-M (4 pigs) or saline (4 pigs). The relative gene expression is given as FC for each experimental group relative to 0 h (FC = 1). These values are then used to compare the gene expression after Matrix-M administration in relation to that after administration of saline.
The immunomodulating complex (iscom) is a formulation in which protein antigens are coincorporate... more The immunomodulating complex (iscom) is a formulation in which protein antigens are coincorporated with glycosylated triterpenoids possessing adjuvant activity in the same particle (Morein et al, 1984). In electron microscopy, the iscoms appear as organized cage-like structures of 30–40 nm in diameter (Ozel et al, 1989). Chemically, iscoms consist of glycosylated triterpenoids from Quillaja saponaria Molina, cholesterol, phospholipids and amphipathic protein antigens (Lovgren and Morein, 1988a). Complexes prepared without protein are called iscom-matrix (matrix) and have the same electron microscopic morphology as iscoms (Ozel et al, 1989). The basic concept of the iscom is to deliver the adjuvant and the protein antigen in the same particle, assuring optimal activation of antigen-presenting cells (APC).
We investigated effects of low titer (Lo) circulating MatAb on protection and immunogenicity of a... more We investigated effects of low titer (Lo) circulating MatAb on protection and immunogenicity of attenuated (Att) human rotavirus (HRV) priming and 2/6-virus-like particle (VLP)-immunostimulating complex (ISCOM) boosting (AttHRV/VLP) or VLP-ISCOM alone vaccines. LoMatAb had both enhancing and suppressing effects on B cell responses, depending on tissue, antibody isotype and vaccine. Differential effects of LoMatAb on IgA responses in different tissues suggest that LoMatAb did not suppress induction of IgA effector and memory B cells but impaired homing of these cells to secondary lymphoid or effector tissues, reducing IgA antibody secreting cells and antibodies at these sites. The AttHRV/VLP vaccine partially overcame LoMatAb suppression, conferred moderate protection against virulent HRV (as measured by reduced viral shedding and diarrhea) and represents a new candidate for rotavirus vaccines for both humans and animals.
Infection with Mycobacterium tuberculosis , the causative agent of tuberculosis (TB), remains one... more Infection with Mycobacterium tuberculosis , the causative agent of tuberculosis (TB), remains one of the leading causes of mortality worldwide. The current “gold standard” vaccine Mycobacterium bovis BCG has a limited efficacy that wanes over time. The development of a vaccine to boost BCG-induced immunity is therefore a highly active area of research. Mucosal administration of vaccines is believed to provide better protection against pathogens, such as M. tuberculosis , that invade the host via mucosal surfaces. In this study we demonstrate that an intranasal vaccine, comprising the antigenic fusion protein Ag85B-ESAT-6 and the mucosal combined adjuvant vector CTA1-DD/ISCOMs, strongly promotes a Th1-specific immune response, dominated by gamma interferon-secreting CD4-positive T cells. Mucosal administration of Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs strongly boosted prior BCG immunity, leading to a highly increased recruitment of antigen-specific cells to the site of infection. Mos...
The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has bee... more The cysteine-rich Pfs48/45 protein, a Plasmodium falciparum sexual stage surface protein, has been advancing as a candidate antigen for a transmission-blocking vaccine (TBV) for malaria. However, Pfs48/45 contains multiple disulfide bonds, that are critical for proper folding and induction of transmission-blocking (TB) antibodies. We have previously shown that R0.6C, a fusion of the 6C domain of Pfs48/45 and a fragment of PfGLURP (R0), expressed in Lactococcus lactis, was properly folded and induced transmission-blocking antibodies. Here we describe the process development and technology transfer of a scalable and reproducible process suitable for R0.6C manufacturing under current Good Manufacturing Practices (cGMP). This process resulted in a final purified yield of 25 mg/L, sufficient for clinical evaluation. A panel of analytical assays for release and stability assessment of R0.6C were developed including HPLC, SDS-PAGE, and immunoblotting with the conformation-dependent TB mAb4...
Immunostimulating complexes were prepared with antigens extracted from tachyzoites of Toxoplasma ... more Immunostimulating complexes were prepared with antigens extracted from tachyzoites of Toxoplasma gondii and were used to immunize mice. The major antigens incorporated into the immunostimulating complexes were the P30 and P22 antigens and an antigen with an approximate molecular weight of 6,000. Other antigens of molecular weights above 30,000 were also present. High antibody titers to T. gondii antigens and a delayed-type hypersensitivity reaction were noted for the immunized mice. Challenge of these mice with tachyzoites injected interperitoneally or with oocysts administered orally resulted in a statistically significant (P < 0.001) conditional probability of survival compared with that of controls. In contrast, the differences between immunized mice and controls challenged with tissue cysts did not attain statistical significance.
B- and T-cell responses have been studied after primary and secondary immunizations of mice with ... more B- and T-cell responses have been studied after primary and secondary immunizations of mice with iscoms containing influenza virus envelope glycoproteins. After primary immunization both B- and T-cell responses were initiated in the draining lymph nodes. T-cells showed peak activity with respect to proliferation and cytokine production after five to eight days and the highest number of IgG secreting cells (IgG-SC) was recorded at day seven. The responses in the spleen developed slowly but were of longer duration. Cytokines produced by spleen cells included high levels of IFN-gamma and IL-2. After a secondary immunization the frequencies of IgG-SC were drastically increased in both LN and spleen, but decreased rapidly with time. At day eight after the secondary immunization high numbers of IgG-SC were detected in the bone marrow. High titres of IgG1 and IgG2a and substantial titres of IgG2b and IgG3 were detected in serum.
ABSTRACTBluetongue virus (BTV) causes bluetongue disease, a vector-borne disease of ruminants. Th... more ABSTRACTBluetongue 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 orEscherichia coliexpression 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 ...
Alignment of B-strains based on hemagglutinin amino acid sequence. B/Bris = B/Brisbane/60/08 [Gen... more Alignment of B-strains based on hemagglutinin amino acid sequence. B/Bris = B/Brisbane/60/08 [GenBank: ACN29380.1], B/Mal = B/Malaysia/2506/04 [GenBank: ACR15732], B/Florida = B/Florida/04/06 [GenBank: ACF54246.1], B/Mass = B/Massachusetts/02/12 [GenBank: AGL06036.1]. Blue bar indicates head domain defined as Asn58 to Glu307. (PDF 156 kb)
Body weight loss and clinical scores following homologous challenges. Mice (6 or 10/group) were i... more Body weight loss and clinical scores following homologous challenges. Mice (6 or 10/group) were immunized once with different doses of seasonal trivalent virosomal influenza vaccine (TVV) with or without Matrix-M™ or PBS and challenged with vaccine-homologous virus strains H1N1 A/Netherlands/602/09, H3N1 A/Perth/16/09 or B/Malaysia/2506/04 and monitored for 21 days for survival, body weight loss and clinical symptoms. Graphs represent mean body weight changes with 95 % confidence interval and median clinical scores with interquartile range. The HA amino acid sequences of H1N1 A/Netherlands/602/09 and H1N1 A/California/07/09 differ by 5 residues (99.5 % homology), those of H3N2 A/Perth/16/09 and H3N2 A/Victoria/210/09 differ by 7 residues (98.8 % homology), and those of B/Malaysia/2506/04 and B/Brisbane/60/08 differ by 5 residues (99.2 % homology) (Additional file 4: Figure S3, Additional file 5: Figure S4, Additional file 6: Figure S5). (PDF 96 kb)
Alignment of H3N2 strains based on hemagglutinin amino acid sequence. H3/Vic = A/Victoria/210/09 ... more Alignment of H3N2 strains based on hemagglutinin amino acid sequence. H3/Vic = A/Victoria/210/09 [GenBank: AFM72883.1], H3/Perth = A/Perth/16/09 [GenBank: ACS71642.1], H3/HK = A/Hong Kong/1/68 [GenBank: ACU79871.1]. Blue bar indicated head domain defined as Cys68 to Cys293. (PDF 146 kb)
Additional file 1. Gene expression in pooled blood samples from pigs administered Matrix-M or sal... more Additional file 1. Gene expression in pooled blood samples from pigs administered Matrix-M or saline. The gene expression was analysed by a qPCR plate array in pooled blood samples collected at 0, 18, 30 h, 2 and 3 days after administration of Matrix-M (4 pigs) or saline (4 pigs). The relative gene expression is given as FC for each experimental group relative to 0 h (FC = 1). These values are then used to compare the gene expression after Matrix-M administration in relation to that after administration of saline.
The immunomodulating complex (iscom) is a formulation in which protein antigens are coincorporate... more The immunomodulating complex (iscom) is a formulation in which protein antigens are coincorporated with glycosylated triterpenoids possessing adjuvant activity in the same particle (Morein et al, 1984). In electron microscopy, the iscoms appear as organized cage-like structures of 30–40 nm in diameter (Ozel et al, 1989). Chemically, iscoms consist of glycosylated triterpenoids from Quillaja saponaria Molina, cholesterol, phospholipids and amphipathic protein antigens (Lovgren and Morein, 1988a). Complexes prepared without protein are called iscom-matrix (matrix) and have the same electron microscopic morphology as iscoms (Ozel et al, 1989). The basic concept of the iscom is to deliver the adjuvant and the protein antigen in the same particle, assuring optimal activation of antigen-presenting cells (APC).
We investigated effects of low titer (Lo) circulating MatAb on protection and immunogenicity of a... more We investigated effects of low titer (Lo) circulating MatAb on protection and immunogenicity of attenuated (Att) human rotavirus (HRV) priming and 2/6-virus-like particle (VLP)-immunostimulating complex (ISCOM) boosting (AttHRV/VLP) or VLP-ISCOM alone vaccines. LoMatAb had both enhancing and suppressing effects on B cell responses, depending on tissue, antibody isotype and vaccine. Differential effects of LoMatAb on IgA responses in different tissues suggest that LoMatAb did not suppress induction of IgA effector and memory B cells but impaired homing of these cells to secondary lymphoid or effector tissues, reducing IgA antibody secreting cells and antibodies at these sites. The AttHRV/VLP vaccine partially overcame LoMatAb suppression, conferred moderate protection against virulent HRV (as measured by reduced viral shedding and diarrhea) and represents a new candidate for rotavirus vaccines for both humans and animals.
Infection with Mycobacterium tuberculosis , the causative agent of tuberculosis (TB), remains one... more Infection with Mycobacterium tuberculosis , the causative agent of tuberculosis (TB), remains one of the leading causes of mortality worldwide. The current “gold standard” vaccine Mycobacterium bovis BCG has a limited efficacy that wanes over time. The development of a vaccine to boost BCG-induced immunity is therefore a highly active area of research. Mucosal administration of vaccines is believed to provide better protection against pathogens, such as M. tuberculosis , that invade the host via mucosal surfaces. In this study we demonstrate that an intranasal vaccine, comprising the antigenic fusion protein Ag85B-ESAT-6 and the mucosal combined adjuvant vector CTA1-DD/ISCOMs, strongly promotes a Th1-specific immune response, dominated by gamma interferon-secreting CD4-positive T cells. Mucosal administration of Ag85B-ESAT-6 mixed with CTA1-DD/ISCOMs strongly boosted prior BCG immunity, leading to a highly increased recruitment of antigen-specific cells to the site of infection. Mos...
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Papers by Karin Lovgren