key: cord-1031940-glpiqzov authors: Serra, Alexandra; Marzo, Nuria; Pons, Berta; Maduell, Pau; López, Maite; Grancha, Salvador title: Characterization of antibodies in human immunoglobulin products from different regions worldwide date: 2021-01-29 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2021.01.034 sha: db007ea0c2d6fba2ae35f76ab59fca88cda03d25 doc_id: 1031940 cord_uid: glpiqzov PURPOSE: The antibody levels against a broad spectrum of pathogens were assessed in commercial intravenous immunoglobulin (IVIG) manufactured from pooled plasma obtained from different global regions. METHODS: Twenty-four IVIG commercial lots from eight manufacturers corresponding to 12 brands were analyzed. The plasma was collected in ten countries/regions. Depending on each pathogen, antibody levels were measured by specific commercial IgG-specific immunoenzymatic assay kits or cell culture neutralization test and guinea-pig skin neutralization test. A principal component analysis was performed. RESULTS: For polio and diphtheria (reference markers by US Authorities) all IVIGs had relevant titers in accordance with reference levels. IVIGs from Canada, Australia and USA were positive for titers against globally distributed pathogens or those under vaccination programs in developed world (parainfluenza, Epstein-Barr, varicella-zoster, influenza-B, parvovirus-B19, measles). IVIG from Taiwan and Hong-Kong showed low antibody titers for these pathogens but high titers forP. aeruginosa. IVIG from India had high titers for pathogens frequently found in developing countries (West Nile, dengue, chikungunya, hepatitis-E, S. pneumoniae). IVIGs from Argentina, Spain, Israel and Czechia showed intermediate antibody concentrations. CONCLUSION: The antibody profile in IVIG was greatly influenced by regional characteristics including climate, vaccination programs and prevalence of pathogens in the different countries and regions. Since the first use of serum antibodies against infectious diseases in the late 19 th century, 1 the use of antibodies has largely been supplanted by antimicrobial agents. However, the generalized use of antibiotics has led to an emerging crisis of drug resistance for many pathogens throughout the world. Therefore, the focus for treating infections is shifting to include alternative therapies. Immunoglobulin (Ig) was successfully used for the first time by the middle of last century to treat a pediatric case of immunodeficiency. 2 Currently, Ig can be used for the treatment of a variety of infections. 3 In 1981, concentrates of purified, functional immunoglobulin G (IgG) known as intravenous immunoglobulin (IVIG), became commercially available in the United States. Recently, the need for readily available therapies to treat COVID-19 during the pandemic has caused investigators to turn their attention back to IVIG as an anti-infective therapy. [4] [5] [6] IVIG preparations are obtained from pooled human plasma of a minimum size of 1000 donors up to 100,000 donors. 7 IVIG products contain specific antibodies resulting in neutralization of a wide range of specific antigens including pathogens. 8 In addition, IVIG preparations from human pooled plasma take advantage of the polyclonal response of every individual donor. IVIG is indicated as a replacement therapy for patients with primary and secondary immunodeficiency. These conditions are characterized by greater susceptibility to infectious diseases due to quantitative or J o u r n a l P r e -p r o o f qualitative antibody deficiencies. 9 In addition to its use in primary and secondary immune deficiencies, IVIG is increasingly used for the treatment of a large number of autoimmune and inflammatory diseases. 10 IVIG efficacy has been widely demonstrated in clinical trials and clinical practice. 11 A minimum potency for antibodies against poliovirus (type 1, 2, or 3), measles, and diphtheria are generally accepted as reference markers for IVIG. 12, 13 Depending on the geographic area where the donor population lives, differences in antibody profiles in manufactured IVIG have been reported. 14-18 For instance, the immune status of the plasma donors, influenced by community vaccination programs and pathogen exposure, may be responsible for the antibody level variability in IgG preparations from different geographical areas. Commercial IVIG manufacturing usually implies the fractionation of large plasma pools from healthy donors. This provides a unique tool to investigate the profile of immunoglobulin G reactivity in these populations. However, there is a lack of systematic studies correlating levels of pathogen-specific antibodies with the donors' geographic origin. To further increase the understanding of the characteristics of IVIG antibody profiles, we determined antibody levels against a broad spectrum of viral and bacterial pathogens in commercial IVIG lots obtained from different geographical areas worldwide. J o u r n a l P r e -p r o o f The aim of this study was to characterize antibodies against a range of pathogens in IVIG samples prepared from pooled plasma obtained in different geographical areas worldwide. These IVIG samples represented countries and populations with diverse vaccination profiles and/or natural exposure to different infectious agents. Twenty-four IVIG commercial lots from eight manufacturers (identified in this study as A through H) corresponding to 12 brands (identified as A.1 through H.4) were analyzed (see Table 1 ). The geographic origin of the 24 IVIG lots included North and South America, Europe and Middle East, and Asia-Pacific regions. More precisely, the ten different countries and territories (at least three for each geographic region) where source plasma was obtained were: Argentina, Australia, Taiwan, Hong Kong, India, Israel, Spain, Czech Republic (Czechia), Unites States of America (USA), and Canada. Details of product and lot distribution by geographic area are shown in Table 1 . Samples from each IVIG lot were analyzed to assess the specific antibody levels against For PV1 and C. diphtheriae toxin, antibody titers were assessed using cell culture neutralization and guinea pig skin neutralization tests, respectively. Although opsonization tests are more difficult to perform and have more variability than conventional binding assays such as ELISA, they provide the best function correlate of protection in assessing vaccine immunogenicity. Additionally, an immunonephelometric assay was performed to measure the levels of residual immunoglobulin M (IgM) 19 in IVIG lots. This could determine possible IgM interactions that could interfere in the interpretation of IgG results. Results were expressed as g/L per product brand (representative of the manufacturing process). All titer analyses were carried out in duplicate. The IVIG antibody titer (mean and standard deviation [SD]) against each pathogen was calculated for each geographical plasma source. Pathogens were grouped as follows. In the first group were those accepted as reference markers according to US regulations. 12 Specifications were set at ≥0.17 ratio sample/CBER Reference at 10% IVIG concentration for PV1 and ≥1.21 AU/mL for C. diphteriae at 10% IVIG concentration. 13 Reference values were not specified for MSLV because titer was determined by ELISA method. The other pathogens were grouped according to their more relevant mode of transmission (vector-borne, water/food-borne, airborne, droplet contact, body fluids contact, and nosocomial/opportunistic). Countries were sorted according to their predominant climatic area (tropical vs. non-tropical) 20 Table 2 summarizes the antibody titers of all pathogens in IVIG preparations from the ten countries and territories worldwide and geographical/climatic areas and their HDI, as well as the total titer per pathogen (mean ± SD). For PV1, antibody titer values were highest in Australia, Hong Kong, Israel, Czechia and Canada (8-10 ratio/g Ig; 3-5 ratio/g Ig for the other regions). Anti-diphtheria antibody results showed a relatively wide range of values among regions. Highest titers were observed in Argentina and Australia (close to >200 AU/g Ig). Titers for IVIG from Taiwan and Czechia were much lower than the rest, averaging 20±0 AU/g Ig for both regions. For MSLV, titer values ranged from 84 IU/g Ig in Hong-Kong to 281±15 IU/g Ig and 330±2 IU/g Ig in Canada and Australia, respectively. In vector-borne (mosquito) disease pathogens (CHIKV, DENV and WNV), the IVIG samples obtained from donors in India showed titer values much higher than the other IVIG, approximately 25-to 50-fold. Differences in the geographical distribution were also observed in antibodies against water/food-borne disease pathogens, specifically for HAV and HEV. Although in both cases the highest titers were again detected in India, HAV showed more variability among countries. Table 3 . Generally, the basal antibody reactivity in a population defines, to some extent, the primary community barrier against pathogen expansion. The antibody profile of a given population responds primarily to two factors: incidence of the pathogens in that geographical region and the specific vaccination programs in their communities. However, in the case of opportunistic or universally distributed pathogens, these factors may not apply. Recently, with the COVID-19 pandemic, we have learned how quickly emergent pathogens can spread worldwide. 22 In any case, the composition of an IVIG product is representative of the antibody profile of the donor population, and thus, a close representation on the immunoreactivity against pathogens in the community. 8, 23 In addition, in contrast with other immunoglobulins, IgG is representative of a stable immunological response, probably the result of a repetitive exposure to the pathogen agent or its vaccine. 8, 24 Large fractionation pools used in IVIG manufacturing offer a unique tool to conduct a prospective analysis of the immunoreactivity against pathogens of for a given population. In general, there is a lack of information on the titers for most antibodies in IVIG products. To the best of our knowledge, this is the most extensive study to date covering the largest number of pathogen-specific antibodies (representative of diseases affecting diverse human populations) and IVIG lots selected on a worldwide basis. We characterized the antibody composition of IVIGs obtained from donors from different geographic areas and also considered their predominant climatic area 20 and their standard of living. 21 Nowadays, antibody titers against PV1, MSLV and C. diphtheriae are required for IVIG batch release. 12, 13 Otherwise evaluation must rely on the published literature, when J o u r n a l P r e -p r o o f available (WNV, 25 CMV, 26-28 ECHV, 16 HAV, 29, 30 HBV, 30 IAV-IAB, 18, 31-34 RUBV, 18, 30 MSLV, 17, 18, 30, 35 RSV, 34 VZV, 30, 35, 36 H. Influenzae, 37 C. tetani, 30, 35 S. pneumoniae, 37 P. aeruginosa, 38 C. diphtheriae, 30, 35 ). Our results for PV1 and C. diphtheriae showed that all IVIGs were in agreement with the antibody titers stated by US authorities. 12, 13 For MSLV, the methodology used was approriate to assess the antibody levels for this pathogen, although it limited the interpretation of the results with respect to reference values. Nevertheless, it is important to clarify that above a minimum that currently has only been set for these three pathogens, antibody titer should not be regarded as an indicator of IVIG efficacy. Globally, the analyzed IVIG samples showed significant levels of antibodies against most of the studied pathogens, although differences associated with the geographic origin of the pooled plasma were observed. This confirms the existence of unique properties in the plasma source of the IVIGs. 14-18, 35 Hence, IVIG lots from Australia and Canada showed the highest titer in 13 of the 24 pathogens studied, most of them including globally distributed viruses (e.g., IBV, PIV, B19, and EBV), 39 and viruses influenced by the local vaccination programs (e.g., MSLV, RUBV, PV1, and MUMV [40] [41] [42] [43] [44] ). Australia and Canada, together with the United States, include communities with the highest standard of living in our list, 21 which showed high titers against (PIV, IBV and EBV). By contrast, Taiwan and Hong Kong lots showed lower titers against most of these pathogens. Interestingly, plasma of Taiwanese subjects has been reported to have low titers against MSLV, RUBV, HAV, HBV and VZV. 30 This being ascribed to immune memory decline or loss. Decreased HAV antibody seroprevalence in Europe and the US has also been described, 29 which is in accordance with our results. Conversely, high titers against J o u r n a l P r e -p r o o f different subtypes of IAV (H5N1, 33 H2N2, 32 and H1N1, 31 ) and VZV 36 [46] [47] [48] The diversity in the strategy of IgG purification among manufacturers could be associated with small variations in the content of IgM in these IVIG products. IgM might be able to interfere with the ELISA kits used for IgG determinations. All IVIG preparations showed very low or undetectable IgM concentrations, with the exception some IVIG lots from manufacturers C and D. However, the elevated antibody titer observed against HEV, WNV, CHIKV and DENV indicated that the possible presence of IgM did not interfere on the IgG measurements carried out in this study. In conclusion, we have presented an extensive study of the specific characteristics of the antibody profiles in IVIG that arise as a result of the different geographic origins of the donor population. All IVIG products are, by definition, effective in clinical practice and all of them have met the required levels for polio and diphtheria antibodies stated by authorities. On the other hand, the antibody profiles were greatly influenced by regional characteristics, including vaccination programs and the prevalence of pathogens in the different regions. Frequent IVIG antibody profiling can be useful in the future to better understand pathogen exposure and immunological barriers in a given population. More research is also needed to understand the potential benefit of expanding vaccination programs to plasma donors from each region or country. The authors are full-time employees of Grifols, a manufacturer of intravenous immunoglobulin products and the funder of the study. The authors are full-time employees of Grifols, a manufacturer of intravenous immunoglobulin products. 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