Vox Sanguinis (2004) 86, 125–129 © 2004 Blackwell Publishing
ORIGINAL PAPER
Intravenous immunoglobulin products contain neutralizing antibodies to vaccinia
Blackwell Publishing, Ltd.
J. C. Goldsmith,1 N. Eller,2 M. Mikolajczyk,2 J. Manischewitz,2 H. Golding2 and D. E. Scott2 1
Immune Deficiency Foundation, Towson, MD, USA Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, MD, USA
2
Background and Objectives Individuals with primary or secondary immune-deficiency diseases may be at risk for vaccinia infection if widespread smallpox-immunization programmes are implemented in the United States of America (USA) for bioterrorism preparedness. The objective of this study was to determine whether commercial immune globulin (intravenous, human) products contain biologically active antibodies to vaccinia that have the potential to protect people, with immune deficiencies, from complications of vaccinia. Materials and Methods Eight currently United States (US)-licensed and two European intravenous immunoglobulin (IVIG) products were tested in a vaccinia plaquereduction neutralization assay. The in vivo activity of five of these lots was assessed in severely immune-deficient mice. Results All tested products contained neutralizing anti-vaccinia activity, in vitro and in vivo.
Received: 10 November 2003, revised 7 January 2004, accepted 10 January 2004
Conclusions The use of IVIG by individuals with inherited or acquired humoral immune deficiencies may provide some protection if they are inadvertently exposed to vaccinia. Key words: anti-vaccinia virus antibodies, immune globulin (intravenous, human), primary immune deficiency, smallpox vaccination.
Introduction Intravenous immunoglobulin (IVIG) products contain antibodies against a wide variety of microorganisms as a result of the immunization history and infectious disease exposures of the individuals donating to the plasma pool from which the IVIG is manufactured. These antibodies determine the proven ability of IVIG products to reduce infection frequency and severity in people with primary and some secondary immune deficiencies. Reports in the scientific and medical literature have detailed the properties of these antibodies in selected IVIG products [1–4]. Although routine smallpox vaccination with vaccinia virus was discontinued in the United States of America
Correspondence: Jonathan C. Goldsmith, MD, 40 West Chesapeake Avenue, Suite 308, Towson, MD 21204, USA E-mail:
[email protected]
(USA) in 1971, it was continued for a number of years in other countries, as well as in the USA, for selected members of the United States (US) armed forces and orthopoxvirus researchers [5]. Vaccination for travellers to smallpox-endemic areas was stopped in 1982 [5]. Orthopoxvirus researchers, and designated emergency ‘first responders’, currently receive smallpox vaccination [6]. In the event of a smallpox outbreak, it is possible that a sizeable portion of the US population would be advised to receive immunization. Although vaccination is relatively contraindicated in immune-deficient people, exposure could still occur by accidental vaccination or inadvertent exposure to a vaccinated person’s skin lesion. People with immune deficiency are at risk for widespread vaccinia infection. Vaccinia immune globulin (VIG) is the only prophylactic or therapeutic biological product for which there is evidence of efficacy in humans [7–11]. VIG is manufactured from the plasma of recently vaccinated donors and contains high amounts of neutralizing antibody against vaccinia.
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We tested the hypothesis that commercial IVIG products might contain anti-vaccinia activity owing to the persistence of anti-vaccinia immunoglobulin in donors who had been vaccinated many years previously [12,13]. Such donors could include older individuals, immigrants to the USA, and US military personnel and dependents. These antibodies, if present, could be important as potential protective agents for vulnerable populations, such as those with primary immune deficiency diseases (PIDDs), in the event of either limited or widespread smallpox vaccination in the US in response to a bioterrorism threat. In the present study, anti-vaccinia immunoglobulin activity in licensed IVIG products and those undergoing license review were evaluated using a plaquereduction neutralization assay (PRNA) and an in vivo assay, to determine the potential for IVIG products to provide neutralizing antibodies in people susceptible to widespread vaccinia infection.
Materials and methods Test and control preparations The positive control preparation was the CBER interim reference vaccinia immune globulin (VIG, lot 1) obtained from the Center for Biologics Evaluation and Research, Food and Drug Administration (CBER/FDA; Rockville, MD, USA) [14]. It is a lyophilized 5% globulin product with a recommended testing range of 1 : 40 to 1 : 20 480, in twofold serial dilutions, in a vaccinia PRNA assay. The negative control preparation was a commercially available 5% albumin product. The test preparations were the licensed IVIG products available in the US market in the spring of 2003, which included: Carimune® (ZLB Bioplasma AG, Glendale, CA, USA), Gamimune® N (Bayer Corporation, Research Triangle Park, NC, USA), Gammagard® S/D (Baxter Healthcare Corporation, Deerfield, IL, USA), Gammar®-P I.V. (Aventis Behring L.L.C., King of Prussia, PA, USA), Iveegam EN (Baxter Healthcare Corporation), Panglobulin® (ZLB Bioplasma AG), Polygam® S/D (Baxter/American Red Cross, Arlington, VA, USA), Venoglobulin®-S (Alpha Therapeutic Corporation, Los Angeles, CA, USA), and two products currently under FDA review: Flebogamma (Instituto Grifols, S.A., Barcelona, Spain) and Octagam® (Octapharma L.L.C., Lachen, Switzerland).
Mice Mice used for in vivo testing were 8–12-week-old severe combined immunodeficiency (SCID) mice (National Cancer Institute, Frederick, MD). All animals were used under a protocol approved by the Intramural Animal Care and Use Committee at the FDA.
Assays In vitro PRNA In brief, test and control preparations were serially diluted in tissue culture medium at the Baxter (Round Lake, IL, USA) research facility and mixed with an equal volume (0·4 ml) of vaccinia virus working stock [New York City Board of Health (NYCBOH) strain] containing ≈ 100–200 plaqueforming units (PFU)/ml. This resulted in 68–87 plaques in control wells. A range of dilutions, from 1 : 2 to 1 : 512 (serial twofold dilutions) for each test preparation, and the recommended range for the positive control, were tested. The mixtures were incubated at room temperature for ≈ 60 min and then inoculated onto host cell monolayers (Vero cells) in triplicate. After adsorption, the inoculum was aspirated and a nutrient agarose overlay was added. Cell culture plates were then placed in a 37 °C CO2 incubator for up to 6 days. Plaques (areas devoid of cell growth) were scored after staining with 0·3% crystal violet in 30% alcohol. A virus control, an equal volume mixture of tissue culture medium and the virus working stock tested concomitantly, were studied to establish the amount of infectious virus input. The antibody titre for a specific sample was expressed as the sample dilution that reduced the number of virus plaques to 50% (determined by linear regression) as compared with the number seen in the virus control. Three lots of each test article were evaluated in the study. Test articles were blinded by an external contractor, GPA International (Newport Beach, CA), according to the standard process of randomization of GPA. Prior to the blinding process, lyophilized products were reconstituted according to the product inserts, and all test articles were normalized to a 5% final protein content, using 0·9% saline or sterile water for injection, by GPA personnel. The coded solutions were then examined for complete dissolution and tested, along with the controls, according to the test system described above. General precautions for handling vaccinia virus-containing samples, and for the disposal of biohazardous waste, were followed.
In vivo protection assay Antibody preparations were randomly selected from the tested lots. Vaccinia virus (NYCBOH-derived, Dryvax strain), 106 PFU in 0·1 ml of tissue culture medium, was mixed with 0·2 ml of 5% IVIG (final dose: 10 mg of each IVIG per injection). These mixtures were incubated at room temperature for 1 h and then injected intraperitoneally (i.p.) into mice. Control mice received virus alone in tissue culture medium. Mice were observed twice daily for pox lesion development and for mortality. A previous experiment with dialysed and undialysed IVIG demonstrated that the presence of the excipients glycine, maltose, glucose or albumin did not affect in vivo neutralization. Six mice per treatment group were tested. All neutralizations and injections were performed on © 2004 Blackwell Publishing Ltd. Vox Sanguinis (2004) 86, 125–129
Antibodies to vaccinia in intravenous immunoglobulin preparations
the same day, on the same batch of mice. Limitations in the number of available mice for these lethality studies precluded the testing of all products and all lots. For prophylaxis experiments, VIG or IVIG were injected i.p. at the indicated doses, 8 h prior to vaccinia challenge with 106 PFU/ml given i.p. Mice were monitored for lethality.
Data analysis In vitro neutralization Plaque readings from the dilutions of each coded sample were subjected to a regression analysis to obtain the 50% plaque-reduction titre (the neutralization antibody titre). Results were submitted to the GPA for decoding. Descriptive statistics (mean and standard deviation) of the neutralization antibody titres for each product were then calculated.
In vivo protection Data were expressed as survival curves and analysed using GRAPHPAD PRISM software. Survival curves for each IVIG were compared with that obtained for virus-only treated mice. Statistical comparison of survival curves was carried out by a logrank test (Mantel-Haenszel test), using GRAPHPAD PRISM version 3·00 for Windows (GraphPad Software, San Diego, CA).
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Table 1 Anti-vaccinia immunoglobulin titres of the immune globulin products tested 50% Neutralization titre (1 ÷ dilution) (mean ± SD)
% Of positive control
Firm
Product name
Alpha Therapeutic Corporation American Red Cross Aventis Behring L.L.C. Baxter Healthcare Corporation Baxter Healthcare Corporation Bayer Corporation Instituto Grifols, S.A.a OctaPharma L.L.C.a ZLB Bioplasma AG ZLB Bioplasma AG Negative control Positive control All products
Venoglobulin®-S 173·0 ± 36·1
7·7
198·3 ± 49·8 72·7 ± 13·6 83·0 ± 33·8
8·9 3·2 3·7
87·3 ± 24·5
3·9
Polygam® SD Gammar®-P, IV IveeGam EN Gammagard® SD Gamimune® N Flebogamma® Octagam® Carimune® Panglobulin® Albumin VIG
130·7 ± 19·5 118·3 ± 31·4 66·0 ± 34·6 178·3 ± 130·8 102·7 ± 19·1 < 2·0 2236·5 121·0 ± 62·3
5·8 5·3 3·0 8·0 4·6 0·0 100·0
a
Currently under FDA review for US licensure.
Results To determine whether in vitro neutralization can be mediated by commercial IVIGs, three lots each of all eight US-licensed IVIG preparations, and three lots each of two preparations that are licensed in Europe, were tested using a PRNA (Table 1). All the immune globulins had some ability to neutralize vaccinia infection. As expected, the IVIGs were not as potent against vaccinia as the VIG preparation, which was manufactured entirely from plasma of donors within a few weeks/months of vaccination. IVIG anti-vaccinia titres were ≈ 3–9% of those observed for VIG. The standard deviations reflect lot-to-lot variation within IVIG product types. To investigate the relevance of in vitro titres further, we tested (using an in vivo neutralization method) selected lots of IVIG for the ability to delay or prevent disease. SCID mice, which lack B and T cells, provide a highly sensitive lethality model for widespread vaccinia infection [15,16]. SCID mice were injected with a mixture of IVIG and vaccinia, as indicated (Fig. 1). Mice were followed-up over time for mortality caused by vaccinia. All IVIG products tested prolonged the survival time of SCID mice. To determine whether pretreatment with IVIG could be effective, one group of mice received IVIG (80 mg) or VIG (40 mg) 8 h prior to vaccinia immunization. Pretreatment with IVIG also prolonged the survival time compared with mice that received virus alone (Fig. 2). Mice given 105 virus mixed with IVIG preparations did not develop disease (data not shown). © 2004 Blackwell Publishing Ltd. Vox Sanguinis (2004) 86, 125–129
Fig. 1 Severe combined immunodeficiency (SCID) mice (six per group) were injected with 106 plaque-forming units (PFU) of vaccinia virus, incubated with 10 mg of the indicated intravenous immunoglobulin (IVIG). P-values (Mantel-Haenszel test) vs. virus alone were as follows: Gamimune, 0·0066; Polygam, 0·0145; Flebogamma, 0·0066; and Venoglobulin, 0·0604. Results are representative of two other similar experiments that used different doses of IVIG products.
Discussion Responses to potential threats of bioterrorism include pre-event immunization recommendations for smallpox vaccination. Some individuals with T-cell defects and hypogammaglobulinaemia can be at risk for serious complications, including death, if exposed to live vaccine agents such as vaccinia [8,17]. For a number of years, a hyperimmune globulin (VIG) was licensed by the FDA for the treatment of complications of smallpox immunization, and now new VIG
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Fig. 2 Severe combined immunodeficiency (SCID) mice (six per group) were injected with the CBER interim reference vaccinia immune globulin (VIG) (40 mg) or intravenous immunoglobulin (IVIG) (Gamimune 80 mg), 8 h prior to the injection of vaccinia (106 plaque-forming units/mouse). P-values (Mantel-Haenszel test) vs. virus alone were as follows: IVIG (Gamimune), 0·0145; and VIVIG, < 0·001.
products are undergoing clinical trials for this indication [18]. VIG has been reported to be clinically useful in individuals with impaired immunity, and patients have been described who survived progressive vaccinia after receiving VIG [8,9]. Individuals with PIDDs have also been cited as potentially benefiting from VIG [7,19]. The presence of antibody to vaccinia in all products tested suggests that currently licensed IVIGs and two products under review for licensure could potentially be useful to treat viraemia associated with intentional or inadvertent smallpox vaccination [20]. Although the titres are low compared with VIG, they are present in all lots of products that were tested and represent 3–9% of the titre found for VIG in the test system. It should be noted that IVIG is given in four- to eightfold higher doses than VIG products, which are administered at ≈ 100 mg/kg. Historically, the recommended prophylactic dose for the prevention of eczema vaccinatum was 50 mg/kg [21]. An effective prophylactic or treatment dose of VIG has never been rigorously defined for any complication of smallpox vaccination. Standard deviations for some products are large, representing lot-to-lot titre variability, perhaps owing to differences in the donor plasma pools used for production. For example, the percentage of older donors, or of military donors, may vary among plasma pools. It is also possible that different IVIG manufacturing methods can influence the proportion of anti-vaccinia immunoglobulin in products. The half-life of these antibodies in immunologically intact individuals and in those with PIDDs, is unknown. However, if the IgG molecules do not have unusual survival characteristics, patients receiving IVIG on a regular periodic basis might have blood levels of anti-vaccinia immunoglobulin that are somewhat higher as a result of accumulation over time. Although a protective dose to prevent or treat viraemia is not known, on average the concentration of products required to inhibit 50% of plaques was 0·413 mg/ml [50 mg/ml diluted 1 : 121 to achieve the
50% infectious dose (ID50)]. A typical dose of IVIG for a patient with PIDD is 400 mg/kg, whereas historically, the prophylactic dose of VIG for vaccinia complications was 50 mg/kg. Although the average ID50 titre of IVIGs is 18-fold lower than for VIG (Table 1), the previously recommended dose of VIG is eightfold less than a typical dose of IVIG for PIDD. These calculations suggest that PIDD patients treated regularly may receive potentially prophylactic levels of vaccinia antibodies. Inadvertent exposures, e.g. accidental skin contact with a vaccinee, may result in a low viral exposure compared with vaccination. In this situation, the ratio of anti-vaccinia immunoglobulin to vaccinia exposure could be more favourable for protection in IVIG-treated immune-deficient individuals. In vivo experiments provide additional assurance of the relevance of neutralization titres. To date, substantial differences among the IVIG products have not been observed in SCID mice (Fig. 1 and data not shown). The SCID mouse assay is capable of detecting fourfold dilutional differences in VIG products, indicating that the IVIGs studied have very similar efficacy (D. Scott et al. unpublished). In vivo assays provide an additional dimension in the assessment of neutralization. In vitro assays, such as PRNAs, detect neutralization of the intracellular mature form of vaccinia (IMV ), which is favoured in culture conditions. In vivo vaccinia infections result in the production of extracellular enveloped virus (EEV), as well as IMV. EEV is believed to be responsible for infection dissemination in vivo [22], and it is neutralized by a different subset of antibodies [23,24]. We speculate that VIG and IVIG may contain some anti-EEV activity, which is responsible for at least some of the observed in vivo effect on SCID mice because injected antibody is present during the onset of viral spread and proliferation. These studies also demonstrate in vivo activity if IVIG or VIG are given to mice prophylactically prior to vaccinia infection (Fig. 2). While a single IVIG result is compared with VIG, additional experiments with other lots and other products invariably demonstrate pre-exposure prophylaxis ability of IVIGs in SCID mice. Although lethality is eventually observed in IVIG-treated mice in all experiments, it should be noted that the SCID mouse model is an extreme example of immune deficiency. Most patients with PIDD and acquired immune deficiencies have some level of endogenous adaptive immunity that would be expected to contribute to clearing of vaccinia infection. The availability of VIG would offer a more potent antibodycontaining material for the treatment of complications of smallpox vaccination. It could also offer day-to-day protection from an accidental contact for an immune-deficient individual if it was administered in an ongoing manner. However, until such an agent completes its manufacture, clinical trials and licensure application, currently available IVIG products may offer some protection from complications © 2004 Blackwell Publishing Ltd. Vox Sanguinis (2004) 86, 125–129
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in individuals with inherited or acquired defects of the immune system.
Acknowledgements The authors wish to thank members of the Immune Deficiency Foundation’s Variola Working Group/Medical Advisory Committee, for their help in planning and reviewing these studies. C.-S. Sun performed the blinded plaque neutralization assays. We also thank Drs J. Cherry and V. Fulginiti for helpful comments, and Dr Christine Anderson of the FDA for maintaining and providing VIVIG reference material. This research was supported, in part, by an unrestricted educational grant from Baxter Healthcare. The FDA work was supported entirely by the US FDA. None of the authors has any financial interest in any of the companies manufacturing the products evaluated in this study. The views of the authors represent scientific opinion and should not be construed as opinion or policy of the US Food and Drug Administration.
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