The moderating role of absorptive capacity and the differential effects of acquisitions and alliances on Big Pharma firms' innovation performance.

In the context of increased pharmaceutical innovation deficits and Big Pharma blockbusters' patent expirations, this paper examines the moderating role of firms' absorptive capacity in external innovation activities of Big Pharma firms. The study indicates a rising interest of Big Pharma in acquisitions of and alliances with biotechnology companies. Unfortunately, this increased interest is not reflected in the number of new drugs generated by Big Pharma. We find that acquisitions of biotech companies have negatively affected Big Pharma firms' innovation performance on average but these acquisitions might have a positive effect at higher levels of acquiring firms' absorptive capacity. Moreover, also acquisitions of pharma companies and alliances with biotech companies only have a positive effect on innovation performance at sufficiently high levels of absorptive capacity. The moderating role of absorptive capacity implicates that a tight integration of internal R&D efforts and (unrelated) external knowledge is crucial for harnessing complementarity effects.

Development and qualification of the parallel line model for the estimation of human influenza haemagglutinin content using the single radial immunodiffusion assay.

Infection with human influenza virus leads to serious respiratory disease. Vaccination is the most common and effective prophylactic measure to prevent influenza. Influenza vaccine manufacturing and release is controlled by the correct determination of the potency-defining haemagglutinin (HA) content. This determination is historically done by single radial immunodiffusion (SRID), which utilizes a statistical slope-ratio model to estimate the actual HA content. In this paper we describe the development and qualification of a parallel line model for analysis of HA quantification by SRID in cell culture-derived whole virus final monovalent and trivalent influenza vaccines. We evaluated plate layout, sample randomization, and validity of data and statistical model. The parallel line model was shown to be robust and reproducible. The precision studies for HA content demonstrated 3.8-5.0% repeatability and 3.8%-7.9% intermediate precision. Furthermore, system suitability criteria were developed to guarantee long-term stability of this assay in a regulated production environment. SRID is fraught with methodological and logistical difficulties and the determination of the HA content requires the acceptance of new and modern release assays, but until that moment, the described parallel line model represents a significant and robust update for the current global influenza vaccine release assay.

Feasibility of single-shot H5N1 influenza vaccine in ferrets, macaques and rabbits.

The feasibility of a single-shot, low-dose vaccination against pandemic influenza was investigated. The immunogenicity and safety of whole inactivated, cell culture-derived H5N1 virus plus CoVaccine HT™ as adjuvant was tested in various animal species. In ferrets, doses of 4.0 and 7.5 µg H5N1 (NIBRG-14; A/Vietnam/1194/04; clade 1) without adjuvant gave low geometric mean haemagglutination inhibition (HI) titres (GMTs) of 21-65 three weeks after intramuscular (IM) injection. The addition of 0.25-4 mg CoVaccine HT™ resulted in GMTs of 255-1470 corresponding with 4-25-fold increases. A second immunization caused GMTs of 8914-23,525 two weeks later, which confirmed strong priming. One out of 8 ferrets injected with antigen alone and 5 out of 32 ferrets injected with adjuvanted H5N1 demonstrated minimal transient, local reactions and two animals immunized with adjuvanted H5N1 exhibited increased body temperature one day after injection. In macaques, 5 µg H5N1 with CoVaccine HT™ or aluminium hydroxide as adjuvant elicited GMTs of 172 and 11, respectively three weeks later. A second immunization resulted in GMTs of 1751 and 123, respectively four weeks later. Analysis of cross-reactivity of antibodies after the first immunization with NIBRG-14 adjuvanted plus CoVaccine HT™ revealed GMTs of 69 against NIBRG-23 (A/turkey/Turkey/1/05; clade 2.2) and 42 against IBCDC-RG-2 (A/Indonesia/5/05-like; clade 2.1.3) while titres with aluminium hydroxide were <10. After the second immunization with CoVaccine HT™, GMT against NIBRG-23 was 599 and against IBCDC-RG-2 254, while those with aluminium hydroxide were 23 and 13, respectively. No local or systemic adverse events were detected in macaques. Safety of 5 µg H5N1 plus 0, 2 or 4 mg CoVaccine HT™ was investigated in a repeated dose study in rabbits. Groups of 6 or 9 male and female animals were immunized IM three times at three week intervals. None of the animals exerted treatment-related adverse reactions during the study or at necropsy 3 or 4 days after treatment. We concluded that a low dose of whole inactivated influenza virus plus CoVaccine HT™ is a promising, single-shot vaccine against pandemic influenza.

[Leading role of genes coding polymerase complex in attenuation of domestic donor viruses for A and B live influenza vaccine].

AIM: To identify the genes that are responsible for attenuation of donor viruses for live influenza vaccine. MATERIALS AND METHODS. Analysis of phenotypical properties of reassortants of wild type A and B influenza viruses with A/Leningrad/134/17/57 (H2N2) (A17) and B/USSR/60/69 (B60) master donor viruses was performed by comparison of their capability to grow at different temperatures in chicken eggs or/and MDCK cells. RESULTS: Ts phenotype of 178 reassortants of A17 with current non-ts influenza A wild type viruses and 33 reassortants of B60 with current non-ts influenza B wild type viruses were evaluated. Reassortants inherited two polymerase genes PB2 and PA or PB 1 from A17 regularly demonstrated ts phenotype. The polymerase PA and PB2 gene segments of B60 independently controlled manifestation of ts phenotype of B60 based reassortants. The other nonpolymerase genes played no role in manifestation of ts phenotype of reassortants A17 and B60 viruses. CONCLUSION: The molecular basis for the development ts phenotype of both A and B influenza vaccine reassortant viruses determined by polymerase genes complex.

Evaluation of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the Apx toxins of Actinobacillus pleuropneumoniae.

The reference strains of the 12 serotypes of Actinobacillus pleuropneumoniae express one or two of three different RTX exotoxins designated Apx I, Apx II and Apx III. The toxins are important virulence factors. In the present study, ELISAs with purified Apx I, Apx II and Apx III, respectively, as antigen were evaluated as candidates for serological diagnosis of Actinobacillus pleuropneumoniae infection in pigs. The pigs were inoculated with biotype 1, serotypes 1-12, and biotype 2, serotype 14, respectively. A strong humoral antibody response was seen to all the three antigens in most pigs irrespective of the serotype used for inoculation. However, titers to the exotoxins secreted by the serotype used for inoculation were generally highest. The results show that toxin proteins of Actinobacillus pleuropneumoniae are antigenically related and that a correlation between serotype and secretion of exotoxin is not revealed serologically in the ELISA test.

Detection of the beta2 toxin gene of Clostridium perfringens in diarrhoeic piglets in The Netherlands and Switzerland.

The two studies presented here were done to determine the prevalence of the alpha, beta, epsilon and enterotoxin genes and the novel beta2 toxin gene of Clostridium perfringens in neonatal or pre-weaned piglets with diarrhoea or necrotic enteritis. All C. perfringens isolates were positive for the alpha and negative for the epsilon and enterotoxin gene, implying that only non-enterotoxigenic type A and C strains were detected. The most important findings were the relatively high prevalence of the beta2 toxin gene in isolates from diarrhoeic piglets in both studies, and, in one of the two studies, absence of strains with only the alpha and beta toxin gene. These data are supportive for the suggestion of a causal relationship of beta2 toxin-producing strains with digestive tract diseases in piglets.

Development of an efficient PCR method for toxin typing of Actinobacillus pleuropneumoniae strains.

A method has been developed which allows the determination of the activator, the structural and the secretion genes of the three toxins ApxI, ApxII and ApxIII in Actinobacillus pleuropneumoniae in only two PCR reactions. The oligonucleotide primers were designed to amplify a significant part of the activator and structural genes apxICA, apxIICA and apxIIICA together in a single PCR reaction giving amplification products which differ in length, in order to be clearly separated by agarose gel electrophoresis. Variations in the apxIA and apxIIIA genes which were found in different serotypes were taken into account in the design of the primers to give a uniform amplification product for both variants of the apxIA and the apxIIIA genes. The secretion genes apxIBD and apxIIIBD are also detected in a single PCR reaction containing two pairs of oligonucleotide primers which yield two differently sized fragments to differentiate between apxIBD and apxIIIBD genes. The reference strains of A. pleuropneumoniae serotypes 1-12 and 104 field strains representing all serotypes obtained from various laboratories worldwide were analysed for their content of apx genes. The two PCR reactions give toxin gene patterns which are characteristic for different groups of serotypes in A. pleuropneumoniae and allow the rapid differentiation of five toxin type groups, group 1 including serotypes 1, 5a, 5b, 9 and 11, group 2 including serotypes 2, 4, 6, 8, group 3 with serotype 3, group 4 with serotype 7 and 12 and group 5 with serotype 10. The method enhances and facilitates differentiation of A. pleuropneumoniae strains for diagnostics and epidemiology and allows the detection of serotypes with atypical toxin patterns.

RTX toxin genotypes and phenotypes in Actinobacillus pleuropneumoniae field strains.

Actinobacillus pleuropneumoniae serotype reference strains and 204 A. pleuropneumoniae field strains representing all 12 serotypes and both biovars 1 and 2, obtained from laboratories from various countries worldwide, were analyzed for the presence of the toxin genes apxIC, apxIA, apxIB, apxID, apxIIC, apxIIA, apxIIIC, apxIIIA, apxIIIB, and apxIIID by DNA-DNA hybridization with specific gene probes. Expression of the toxins ApxI, ApxII, and ApxIII was assessed by immunoblot analysis with monoclonal antibodies. The results show that the patterns of apx genes and those of the expressed Apx toxins in biovar 1 field strains are the same as those of the genes and toxins of corresponding serotype reference strain. We found only three strains which had certain apx genes missing compared with the genes in their serotype reference strains. Analysis of the expression of the three toxins showed that nearly all strains expressed their apx genes and produced the same Apx toxins as their serotype reference strain. We found only one strain that did not produce ApxI, although it contained the apxICABD genes, and one strain which did not express ApxII but which contained apxIICA. Several field strains which initially showed that their serotype did not correspond to the apx gene profile of the reference strain and which had an unexpected virulence for the given serotype revealed that their initial serotyping was erroneous. We show that the apx gene profiles are inherent to a given serotype. The method cannot differentiate between all 12 serotypes. However, it allowed us to distinguish five groups of toxin gene patterns which showed pathological, toxicological, and epidemiological significance. None of the biovar 2 strains contained apxIII genes. The apxI and apxII genes in the biovar 2 strains, however, were the same as those found in the serotype reference strains of biovar 1.

Identification of F11 fimbriae on chicken Escherichia coli strains.

Fimbriae were purified from Escherichia coli strains isolated from chickens with septicemia or colibacillosis. When grown on solid media, these strains expressed fimbriae with an apparent subunit molecular mass of 18 kDa. Morphological, biochemical, serological, functional, and molecular characterization revealed that these 18-kDa fimbriae are identical to F11 fimbriae, which were previously found to be involved in the pathogenesis of human urinary tract infection. Screening of a large strain collection showed that 78% of chicken E. coli strains expressed F11 fimbriae, whereas this percentage increased to 96% when the only strains taken into account were those with the serotypes most commonly encountered in avian colibacillosis (O1:K1, O2:K1, O35, and O78:K80). The prevalence of F11 fimbrial expression appeared to be independent of the country of isolation of the strains, except for the United States, where the prevalence seemed higher. Expression of F11 fimbriae by chicken E. coli strains could not be correlated with adherence to chicken tracheal or pharyngeal cells.

Surface antigens from Escherichia coli O2 and O78 strains of avian origin.

Fimbriae from O2 and O78 virulent strains of avian Escherichia coli were compared with type 1A fimbriae with regard to the apparent molecular weights of their subunits and their antigenic relationships. Under static broth culture conditions, most O78 strains expressed fimbriae closely related to those of type 1A. Under the same culture conditions, another type of fimbriae, sharing some common properties with type 1A fimbriae, was observed only on O2 strains; however, these fimbriae differed from type 1A fimbriae in the apparent molecular weights of their subunits and in the expression of specific epitopes. They were called type 1-like fimbriae. Homologies in lipopolysaccharide and outer membrane protein profiles were also demonstrated among the strains expressing type 1-like fimbriae, which suggests the existence of a clonal relationship among O2:K1 avian E. coli strains. The O78 strains studied did not appear to be clonally related.

Fimbrial serotypes of Escherichia coli strains isolated from extra-intestinal infections.

Strains of Escherichia coli isolated from urinary tract infections and meningitis were characterised by their O:K serotype, haemolysin production, mannose-resistant haemagglutination, and the serotype of the P-fimbriae. The P-fimbriae of 71% of the mannose-resistant haemagglutination-positive strains from urinary tract infection and meningitis could be determined with specific monoclonal antibodies. Many strains expressed multiple P-fimbriae serotypes. The serotypes of P-fimbriae found most frequently among mannose-resistant haemagglutination-positive E. coli from urinary tract infections were the F11, F7 and F8 fimbriae, and among meningitic strains, F11, F8 and F9 fimbriae. The expression of certain F-serotypes did not correlate with O:K antigens.

Isolation and characterisation of dog uropathogenic Escherichia coli strains and their fimbriae.

A number of Escherichia coli strains have been isolated from dogs with urinary tract infections. These strains have been characterised with respect to their O, K, H, and fimbrial antigens, colicin production, antibiotic resistance, plasmid content and their ability to haemagglutinate erythrocytes from various species. Crossed immunoelectrophoresis of fimbrial extracts, as well as the reaction of partly purified fimbriae of a number of these strains with monoclonal antibodies revealed homology or a strong crossreaction with an F12 fimbrial subunit protein of human uropathogenic E. coli strains. Unlike human F12 fimbriae producing strains, the dog isolates did agglutinate dog erythrocytes in the presence of D-mannose but not human erythrocytes, indicating that the adhesin carried by these strains is different from the adhesin on fimbriae of human uropathogenic E. coli. Similar indications were obtained from experiments with latex beads coated with the receptor for P-fimbriae. These beads were agglutinated by Escherichia coli strains from human urinary tract infections, but not by the dog isolates described here. Preliminary adhesion experiments of human and dog Escherichia coli to human bladder epithelial and canine kidney epithelial cells also showed differences in adhesion depending on the origin of the strain tested.

Serological response to the P fimbriae of uropathogenic Escherichia coli in pyelonephritis.

Uropathogenic Escherichia coli strains isolated from four patients with pyelonephritis were characterized by their O:K serotype, hemolysin production, mannose-resistant hemagglutination, and the serotype of the P fimbriae. These P fimbriae were serotyped with specific monoclonal antibodies. Serum samples from the patients were analyzed for the presence of specific antibodies to the P fimbriae. In all cases antifimbrial antibodies were found, strongly suggesting that these P fimbriae are expressed in vivo. However, the antibodies in the patient sera were not able to inhibit the mannose-resistant hemagglutination. This finding suggests that these antibodies react with the fimbrial components and not with the minor components which are responsible for adhesion.

Monoclonal antibodies raised against Pap fimbriae recognize minor component(s) involved in receptor binding.

Pap fimbriae were purified from a recombinant strain and used for the production of monoclonal antibodies (MAbs). These MAbs were screened in a fimbriae ELISA with eight different P fimbriae as well as 1A and 1C fimbriae. Five MAbs were specific for Pap fimbriae whereas one MAb did react with Pap, F7(2) and F11 fimbriae. Previously, we described two F11 MAbs which also reacted with Pap, F7(2) and F11 fimbriae. In a whole bacteria ELISA it was shown that the MAbs, which recognized Pap, F7(2) and F11 fimbriae, reacted with recombinant strains which did not express Pap or F11 fimbriae, but still expressed the globoside binding properties. Not one of the five MAbs which are specific for Pap fimbriae reacted with these globoside binding recombinant strains. In a haemagglutination and adherence assay it was shown that only the MAbs which recognized the Pap, F7(2) and F11 fimbriae inhibited the adhesive properties of the globoside binding recombinant strain. Therefore it is concluded that in the present study MAbs are presented which recognize the minor components responsible for adhesion.

Transfer of enhanced resistance against Listeria monocytogenes induced with ribosomal RNA and the adjuvant dimethyldioctadecylammonium bromide.

In this study we investigated the mechanism of enhanced resistance against Listeria monocytogenes induced with Listeria ribosomal RNA and the adjuvant dimethyldioctadecylammonium bromide (DDA). Mice immunized with DDA alone (which were not protected against Listeria-infection) were used as negative controls. Mice immunized with RNA plus DDA were found to have an increased capacity to mobilize polymorphonuclear leukocytes (PMNs) and macrophages to the inflamed peritoneal cavity compared to mice immunized with adjuvant alone. Intraperitoneal (i.p.) inflammation was induced by injection of the sterile irritant proteose peptone. The protective capacity of various cell-populations was investigated by i.p. transfer of cells to normal recipient mice and concomitant challenge of recipient animals with a lethal dose of viable Listeria. Inflammatory PMNs as well as inflammatory macrophages from mice immunized with RNA plus DDA protected recipient animals against listeriosis whereas cells from mice immunized with DDA alone failed to do so. Therefore, enhanced mobilization as well as activation of PMNs and macrophages may have contributed to the expression of protection against L. monocytogenes induced with RNA plus DNA.

T-cell-independent macrophage activation in mice induced with rRNA from Listeria monocytogenes and dimethyldioctadecylammonium bromide.

Purified rRNA from Listeria monocytogenes or Pseudomonas aeruginosa injected in combination with dimethyldioctadecylammonium bromide (DDA), protects mice nonspecifically against a lethal challenge of various extra- and intracellular bacteria. In the present study vaccination of BALB/c as well as C57BL/Ka mice with listerial RNA-DDA resulted in activation of fixed-tissue macrophages, as measured by an enhanced in vivo L. monocytogenes killing in spleen and liver. Evidence was found that macrophage activation by vaccination with rRNA-DDA occurred by a T-cell-independent mechanism. Treatment of mice with cyclosporin A had no effect on the enhanced L. monocytogenes killing induced with RNA-DDA; in vitro exposure of RNA-DDA to spleen cell cultures did not give rise to any lymphocyte proliferation. No evidence could be found for a possible adjuvant activity for RNA-DDA in cellular responses; in fact, RNA-DDA had an inhibitory effect on lymphocyte proliferative responses to Listeria antigen and to concanavalin A.

Monoclonal antibodies for serotyping the P fimbriae of uropathogenic Escherichia coli.

Monoclonal antibodies (MAbs) against seven serologically different P fimbriae (F7(1), F7(2), F8, F9, F11, F12, and F13) of uropathogenic Escherichia coli were tested for their ability to detect the P fimbriae on wild-type strains. In a plate agglutination test the MABs could detect the fimbriae on strains which expressed cloned fimbriae but not on wild-type strains. In a coagglutination test and in a whole-bacterium enzyme-linked immunosorbent assay the MAbs recognized the fimbriae on strains with cloned fimbriae and on wild-type strains. However, the coagglutination test has some disadvantages: only immunoglobulin G MAbs can be used, and the results cannot be read in an objective way. From these results, we concluded that the whole-bacterium enzyme-linked immunosorbent assay is the most convenient method for the determination of P fimbriae on wild-type E. coli strains. With this fast and easy method it is possible to do epidemiological studies on the distribution of P fimbriae among clinical isolates of uropathogenic E. coli and to extend the O:K:H serotype with the F serotype.

Dissociation between enhanced resistance and delayed hypersensitivity induced with subcellular preparations from Listeria monocytogenes and the adjuvant dimethyl-dioctadecyl-ammonium bromide.

In this study we investigated the relation between enhanced resistance and delayed hypersensitivity (DH) induced with subcellular preparations from Listeria monocytogenes and the adjuvant dimethyldioctadecylammonium bromide (DDA). Ribosomal RNA as well as cell envelope fragments (fraction I) protected mice against lethal Listeria infection. However, only fraction I induced DH against killed Listeria. For the induction of protection with fraction I or RNA as well as for the induction of DH with fraction I, preparations had to be administered in combination with DDA. Fraction I elicited a DH response in mice immunized with viable Listeria, but RNA did not. These observations pointed to a dissociation between DH and enhanced resistance induced with RNA, and to a dissociation between fraction I and RNA with respect to their ability to induce or elicit DH. Also DH and enhanced resistance induced with fraction I could be dissociated. Intracutaneous administration of fraction I induced high levels of DH without concomitant induction of protection against lethal challenge with Listeria. On the other hand, intraperitoneal administration of fraction I fully protected mice against lethal infection, but only induced a moderate DH response. DH induced with fraction I was largely specific, whereas enhance resistance induced with this preparation was nonspecific. Finally, proteinase K-sensitive proteins were found to be essential for the induction of DH but not for the induction of protection with fraction I.

Monoclonal antibodies that recognize the P fimbriae F71, F72, F9, and F11 from uropathogenic Escherichia coli.

The Escherichia coli P fimbriae F71, F72, F9, and F11 from four cloned strains were purified, and polyclonal antisera were raised in rabbits. Cross-reactions of these antisera with eight different cloned and purified fimbriae were measured in an enzyme-linked immunosorbent assay. These antisera showed a reaction with the homologous fimbriae and also with most heterologous fimbriae. Monoclonal antibodies (MAbs) directed against the same four native fimbriae were produced by the fusion of spleen cells from immunized BALB/c mice with SP2/0 myeloma cells. The resulting four series of MAbs were also screened in an enzyme-linked immunosorbent assay with eight different cloned and purified fimbriae. Four different F71 hybridomas produced MAbs which recognized only epitopes on F71 fimbriae. Two F72 MAbs recognized epitopes on F72 and F9 fimbriae, whereas another F72 MAb recognized an epitope on only F72 fimbriae. Three MAbs raised against F9 reacted only with epitopes on F9 fimbriae. Six MAbs against F11 fimbriae could be divided into two groups: on the one hand two MAbs recognizing F11, pyelonephritis-associated pilus, Pap, and F72 fimbriae and on the other hand four MAbs recognizing F11 and "Clegg" fimbriae. None of the MAbs reacted with 1A or 1C fimbriae. In a hemagglutination inhibition assay it was shown that none of the MAbs produced inhibited the adhesive properties of homologous cloned strains.

RNase-sensitive and RNase-insensitive protective components isolated from Listeria monocytogenes.

Crude ribosomes were isolated from Listeria monocytogenes serotype 4b and separated into two fractions by molecular sieve chromatography. Chemical analysis indicated that fraction I contained cell envelope components while fraction II contained the ribosomes. Both fractions protected mice against Listeria, but only in combination with the adjuvant dimethyldioctadecylammonium bromide (DDA). RNase-treatment, but not proteinase K-treatment destroyed the protective properties of fraction II, and RNA purified from fraction II also induced protection. Protection induced by fraction I was not affected by either RNase- or proteinase K-treatment. Both subcutaneous and intraperitoneal, but not intravenous administration of fraction I, fraction II, or purified RNA induced significant protection against intraperitoneal infection, the intraperitoneal route of administration being the most effective. All preparations induced high levels of protection 3 to 7 days after administration, but protection was already decreased after 14 days. Protection induced with RNA appeared to be biphasic, because it also protected mice 1 day, but not 2 days after administration. Protection induced with both fraction I and RNA was at least in part non-specific, because both preparations also protected mice against L. monocytogenes serotype 3, Streptococcus pneumoniae and Pseudomonas aeruginosa. Results are discussed in relation to previous work with analogous preparations from P. aeruginosa.