West Nile Virus Neutralization by US Plasma-Derived Immunoglobulin Products

Abstract

The 1999 introduction of West Nile virus (WNV) into the United States has resulted in the largest epidemic of arboviral illness in the Western Hemisphere, with an estimated 2.5 million cases of mostly asymptomatic human infections since then. As a consequence, an increasing occurrence of WNV antibodies in plasma collected in the United States, and thus in intravenous immunoglobulin (IVIG) products, can be expected. Using an in vitro assay to investigate antibody function, rather then presence, almost 1000-fold differences in neutralization capacity were demonstrated between individual IVIG lots. In a mouse model of lethal WNV infection, treatment with IVIG of a higher WNV antibody titer protected recipients, whereas mice treated with control IVIG died. IVIG lots with higher WNV antibody titers would seem to be desirable for substitution therapy for people with immunodeficiencies.

West Nile virus (WNV), a mosquito-transmitted flavivirus first isolated from a blood specimen obtained in Africa in the late 1930s [1], was newly introduced into the United States in 1999 [2]. Although the exact circumstances of virus introduction remain unclear, a significant similarity between isolates from Israel (Isr98) and New York (NY99) suggests that the virus now circulating in the United States was originally imported from Israel [3].

During the years since its initial introduction, WNV has caused annual epidemics of significant magnitude in the United States, with an estimated cumulative total of 2.5 million human infections between 1999 and 2006 [4]. The 1999 event thus marked the beginning of an epidemiologically rare process-the exposure of an entirely naive population to a now widely prevalent virus.

Because only a minority [5] of reported human WNV infections cause any clinical symptoms and even fewer cause a lethal outcome, an increasing proportion of the US population can be estimated to have been infected with WNV at some point. In response to the WNV infection, these individuals carry WNV antibodies, which are expected to be long-lived [6]. As a consequence, an increasing WNV seroprevalence should have developed in the US population, from 0 in early 1999 to the present level.

Plasma of US origin is the globally predominant source for fractionation into plasma derivatives, including immunoglobulin products. These products are used, among other indications, for intravenous immunoglobulin (IVIG) substitution treatment in people with immunodeficiencies. Because these people do not have a functioning immune defense, they critically depend on the presence of a broad variety of antibodies in the immunoglobulin products with which they are treated, to ensure protection against all the possible infectious agents they may encounter [7].

Since its initial introduction of the WNV into the United States in 1999, infection with WNV has been a certain concern for people living there [8], despite the typically rather benign course of illness in otherwise healthy individuals. For people with immune deficiencies, however, the consequences of WNV infection could potentially be much more severe [9], which likely contributes to a more significant level of concern expressed by this particular group of individuals [10].

The increasing WNV seroprevalence in the US population would conceivably also result in increasing levels of WNV antibody in immunoglobulin products derived from plasma collected from this population. By contrast, WNV infections in Europe have been very rare up to now, which makes the presence of WNV antibody in IVIG products derived from European (EU) plasma unlikely. Because passively transferred antibodies against various flaviviruses, including WNV (summarized in [11]), have been shown to afford protection against subsequent virus infection, immunoglobulin products that contain higher levels of WNV antibodies might be protective against WNV infection. The present investigation sought to determine the levels of WNV antibodies in immunoglobulin products manufactured from US plasma using a functional in vitro assay (i.e., neutralization of virus infectivity) and to correlate the presence of these WNV antibodies with potential protection against WNV infection in a small animal model of WNV infection.

Materials and Methods

Virus and cells. WNV (isolate 385–99, obtained from the liver of a snowy owl found dead in New York in 1999; provided by Dr. Robert E. Shope, University of Texas Medical Branch, Galveston [12]) was propagated in and titrated on Vero cells (ATCC CCL 81). Samples containing WNV were titrated by TCID50 assay using serial 0.5-log dilutions of samples. Cells were incubated for 7 days with virus that contained samples before any virus-induced cytopathic effect was evaluated. Virus concentrations were calculated according to the Poisson distribution and are expressed as log₁₀ TCID50 per milliliter.

WNV neutralization assay. Equal volumes containing a target of 10,000 or 4 log₁₀ TCID₅₀ of WNV and test article (i.e., WNV antibody-containing or control solutions) were mixed and incubated with gentle agitation for 1 h at room temperature. The reaction mixture was subsequently titrated immediately as described above. WNV neutralization was calculated as the difference between the titer determined for the input virus suspension, compared with the reading obtained for the identical virus preparation after incubation with the test or a control article, corrected for the dilution of virus, and expressed as log₁₀ TCID₅₀. As a positive control, the WNV monoclonal antibody 7H2 (81–002; Invitrogen) was used [13].

As test articles, a 10% liquid IVIG preparation (Gammagard Liquid [Baxter Healthcare][Baxter]) was diluted 1:10, or a 5% lyophilized IVIG reconstituted in accordance with the manufacturer's instructions (Gammagard SD; Baxter Healthcare) was diluted 1:5. Several lots were analyzed and were manufactured exclusively from plasma collected either in the United States or Europe.

In addition, serum obtained from US blood donors during convalescence from polymerase chain reaction (PCR)-confirmed WNV infection (provided by Dr. Susan L. Stramer, American Red Cross, Gaithersburg, MD) were used as test articles, undiluted and after heat inactivation for 10 min at 56C. Each specimen was tested at least in triplicate and typically on 3 different days. Differences in WNV neutralization titer between individual test articles were evaluated by unpaired t test, and data are expressed as means ± SEs.

Annualized incidence of WNV infection in individual states. The annualized incidence of symptomatic WNV infections in individual states was calculated by dividing the cumulative number of cases that were reported in the respective state between 1999 and 2006 by the states’s population according to the US Census Bureau [14] and the number of years evaluated.

Protection against WNV infection. Female BALBmice (Charles River Laboratories), 6–8 weeks old, were used for all experiments. Mice were infected with WNV by subcutaneous (sc) injection of 0.2 mL of WNV stock, diluted to contain 100,000 or 5 log₁₀ TCID₅₀ WNV, on the back. Survival of WNV-challenged mice was recorded for 28 days. Where indicated, mice also received an sc injection of 0.2 mL of US plasma- derived (US 21; neutralization titer 2.4 ± 0.1) or EU plasma- derived (EU 1; neutralization titer 0.1 ± 0.2) IVIG products (diluted 1:10 in Tris-buffered saline) or a dilutionbuffercontrol 2 h before WNV infection on the contralateral side of the back. Survival curves were created by the Kaplan-Meier method and compared by log-rank test. All animal experiments were executed in compliance with the Austrian Animal Experiments Act (No. 5011989).

Results

WNV neutralization by IVIG. Significant differences were observed ( P = .0029) (figure 1) when we compared WNV neutralization by IVIG (Gammagard Liquid), manufactured either from EU-or US-sourced plasma. Whereas product lots manufactured from EU plasma showed no neutralization effect (mean ± SE, 0.1 ± 0.0; n = 3), the values obtained for lots produced from US plasma averaged at 1.4 0.1 (n = 32). Also, however, substantial variability between the US plasma- derived lots tested became apparent, with neutralization ranging from only 0.3 to as high as 2.9 among individual lots (i.e., a difference of 2.6 log₁₀ or ∼400-fold).

Figure 1.

West Nile virus (WNV) neutralization by European Union (EU) or US plasma-derived intravenous immunoglobulin (IVIG). Results shown are means (black bars) ± SEs (white bars) of at least 3 independent experiments, typically executed on 3 different days. US lots to the right of the dashed line are statistically equivalent to US lot 21 marked by the arrow, which was used for the in vivo protection assay. Differences in WNV neutralization titer between individual test articles were evaluated by unpaired t test.

We tested another IVIG preparation, produced using a different manufacturing process (Gammagard SD) and also derived from EU or US plasma, and essentially similar results were obtained (mean ± SE, 0.0 ± 0.0, n = 4 [EU]; 1.0 ± 0.2, n = 10 [US]; P = .0083), which indicates that the neutralizing effect on WNV is not impaired by the purification and viral reduction steps used in both processes. There was, however, no significant difference in WNV neutralization titer between IVIG product lots manufactured from plasma either obtained by plasmapheresis (i.e., source plasma) or plasma recovered from whole blood donations (i.e., recovered plasma) of US origin (source vs. recovered [Gammagard Liquid KIOVIG], P p .15 ).

WNV neutralization by human convalescent serum. To determine the reason for the significant differences in WNV neutralization titers that were observed among US plasma- derived IVIG product lots, the WNV neutralization capacity was tested of serum samples obtained from blood donors with PCR-confirmed WNV infection. The mean neutralization titers of these convalescent serum samples were significantly (P = .0007) higher than those earlier found for US plasma-derived IVIG products, with a mean ± SE average titer of 2.8 ± 0.1 (n = 4). In other words, these individual serum specimens had a WNV neutralization titer of ⩾1.4 than the average of all IVIG lots earlier tested (i.e., a minimum of 25-fold higher).

Annualized WNV incidence in individual states. Although plasma pools for manufacturing are composed of thousands of individual donations, larger shipments of plasma collected in a geographically limited region may represent a dominant proportion of any given plasma manufacturing pool. Geographic differences in the cumulative WNV incidence between 1999 and 2006 may thus likely be a factor that could influence the WNV antibody titer of individual pools. To evaluate the contribution of this factor, the annualized WNV incidence of each state in the United States was calculated for the period between 1999 and 2006 (i.e., the time since WNV first entered the United States). These incidence rates differed by >100-fold among states, with substantial east-to-west variation (figure 2).

Figure 2.

Cumulative annualized West Nile virus (WNV) incidence per 100,000 population in the United States. Shown are symptomatic cases of WNV that occurred between 1999 and 2006 in respective US states, sorted by capital from east to west, divided by population and years of WNV endemicity.

Protection against WNV infection. For flaviviruses, the infection of mice is a well-established model to investigate the mechanisms and the efficacy of immune protection against infection with these viruses [15-17]. The specific WNV infection used (i.e., sc challenge with 100,000 TCID₅₀ or ~800 LD₅₀ doses) resulted in survival of only 10% of the control mice (mean of 2 experiments, with 10 micein each series; figure 3).

Figure 3.

Survival of mice infected with West Nile virus (WNV). Mice were treated with either intravenous immunoglobulin (IVIG) derived from European Union (EU) or US plasma samples (10 mice) or control samples (Tris-buffered saline; 5 mice), 2 h before WNV challenge (10⁵ TCID₅₀ WNV, subcutaneously). Survival was monitored for 28 days, and results are means of 2 independent experiments. Survival curves were created by the Kaplan-Meier method and compared by log-rank test.

Treatment of mice 2 h before WNV challenge with 0.2 mL of 10% IVIG manufactured from EU plasma and diluted 1:10, for a preparation of the typical WNV neutralization titer for this geographic origin ( 0.1 ± 0.1; n = 24), did not significantly change this outcome (20% survivors, P = .26 vs. buffer control) (figure 3). When mice were instead treated with US plasma-derived 10% Gammagard Liquidwith a relatively higher WNV neutralization titer ( 2.4 ± 0.1, n = 20), however, 90% of mice survived this otherwise mostly lethal challenge ( P < .0001 vs. control) (figure 3).

Discussion

Since the first US WNV outbreak in 1999, with initially only 62 cases reported in the greater New York area [18], WNV has caused annual epidemics of ever-increasing magnitude and has spread across the entire North American continent from east to west. With 23,886 cases of infection [19] reported through the end of 2006, this spread of WNV represents the largest outbreak of any arthropodborne viral disease ever recorded in the United States [18]. Taking into account that ~80% of all human WNV infections remain asymptomatic [20, 21] and that <1% of infected persons develop neuroinvasive disease, it is assumed that the majority of cases would not be captured by the US national surveillance system for arthropodborne virus diseases (ArboNET) and that the total number of persons ever infected with WNV in the United States is much higher than the number of reported cases. As other researchers have suggested [4], the 9794 cases of neuroinvasive WNV infection that have been reported in the United States between 1999 and 2006 [19] would correspond to ~2.5 million human infections that could have occurred in the United States to date.

On the basis of this estimate, past infection with WNV would be rather prevalent in the US population, approaching almost 1% of the entire population. Therefore, plasma collected in the United States, the largest source of plasma for fractionation into plasma derivatives in the world, should contain WNV antibodies, potentially in clinically meaningful concentrations.

In the present study, the concentration of functional WNV neutralizing antibodies-that is, those neutralizing the virus versus only binding it (e.g., as determined by ELISA)-was determined for several lots of 2 different IVIG products, Gammagard Liquidand Gammagard SD. For both products, lots were analyzed that were produced exclusively from either plasma collected in Europe or the United States. As can be seen in figure 1, US plasma-derived IVIG products contained WNV neutralizing antibodies in concentrations significantly higher than those in identical products manufactured from EU plasma.

Because all human clinical cases of WNV infection have been caused by lineage 1 virus isolates, including the large number reported in the United States and the fewer cases in Europe, cross-reactivity among these viruses can safely be assumed [2].

In Europe, however, the overall number of cases has remained rather limited, compared with those observed after the introduction of the WNV into the United States in 1999. Specifically, even during the largest WNV outbreak, 453 reported human cases in Romania in 1996 [22], the calculated incidence that year would have been 1.9 cases100,000 population-an order of magnitude lower than the annualized incidence of some of US states, where WNV has been active for several years now [23]. In EU countries that are more active in plasma sourcing (e.g., Germany and Austria), cases of human WNV infection have not occurred [20]. The lack of any WNV neutralization effect by EU plasma-derived IVIG, in contrast to the very significant levels of neutralization as measured for certain US plasma-derived IVIG lots, is thus entirely plausible.

The variation in WNV neutralization titers within US plasma- derived IVIG products may be somewhat less intuitively clear. However, the cumulative effect of (1) plasma donations collected from persons after convalescence from WNV infection having a very high WNV neutralization capacity and (2) the potential predominance of plasma from a certain geographic region for which the cumulative number of WNV infections being significantly above average (figure 23) within a given plasma manufacturing pool can result in rather dramatically different WNV titers in IVIG products. As can be seen in figure 1, the WNV neutralization values determined within the present study covered a range of almost 3 orders of magnitude.

Because WNV and St. Louis encephalitis virus (SLEV) belong to the same serocomplex within the Flavivirus genus, the occurrence of SLEV infections in the United States might potentially also have influenced the WNV neutralization titers determined in the present study. However, the cumulative number of cases of SLEV infection between 1964 and 2005 was only 4651 [24], compared with a total of 23,886 cases of WNV infection within the past 8 years-that is, 5-fold more cases within a 5-fold shorter period of time. Also, the advanced age of those individuals infected earlier with SLEV would disqualify a larger proportion of them as plasma donors, making any significant contribution of SLEV antibodies to WNV neutralization titers determined in the present study exceedingly unlikely.

Although the demonstration of virus neutralization in a functional in vitro assay can provide good evidence for a corresponding effect in vivo, more convincing evidence for a potentially beneficial clinical effect would be provided by the direct demonstration of efficacy in vivo-that is, the demonstration of protection against WNV infection. Mice are susceptible to infection with other flaviviruses [16], including WNV infection [15], and are thus a suitable and widely used preclinical model. In the present study, sc injection of mice was chosen for infection with WNV, because this route is anatomically more similar to natural infection via mosquito bites.

Compared with substitution therapy of people with immunodeficiencies, mice were treated with doses of IVIG at the lower end of those typically used in our investigation. Specifically, mice were treated with ~100 mg, whereas, typically, infusions of 400–600 mgonce a month are used for substitution therapy [25]. When subsequently challenged with a dose of WNV, only 10% of control mice survived, and mice treated with IVIG lot manufactured from EU plasma of insignificant in vitro WNV neutralization titer were not protected. By contrast, 90% of mice treated with the higher WNV neutralization titer IVIG lots manufactured from US plasma survived the WNV challenge.

In principle, this effect is not surprising, because protection against flavivirus infection by immunoglobulin preparations that neutralize the corresponding virus in vitro has been reported for another flavivirus, tickborne encephalitis virus [16], and for WNV [15]. One group has even reported that human IVIG from Israeli plasma (i.e., obtained from a region where WNV epidemics have occurred since the 1950s) contained sufficient WNV antibody to protect mice against virus challenge. A similar product from US plasma was ineffective, “as the US population did not begin to develop immunity to WNV until 1999” (p. 9) [17].

Meanwhile, as a consequence of the rather wide prevalence of WNV in the United States, the present investigation determined high WNV neutralization titers now also in certain IVIG lots produced from US plasma. Given that IVIG with a higher WNV antibody titer was shown to confer protection against WNV challenge in an animal model, material with this potentially clinically desirable characteristic might be more widely available than has been appreciated. If WNV has established endemicity in the United States, one might even assume that WNV neutralization titers of IVIG from US plasma might continue to increase, qualifying an increasing number of IVIG lots for possible use in protecting humans against WNV infection.

Because ~1% of the US population has been infected with WNV so far, the concerns of the immunodeficiency community [10]-people significantly more vulnerable to any infection that is not effectively prevented by their IVIG substitution treat-ment-are well understood. In fact, this group might, if not adequately protected by their treatment regimen, be at risk of developing a more severe course of disease because of their underlying medical condition, although supporting clinical evidence to date has been limited to reports of severe WNV infection in immunosuppressed transplant recipients [26].

Qualifying IVIG lots for WNV neutralization titer might offer an opportunity to improve current treatment regimens, such that specific higher titer lots would preferentially be used for IVIG substitution therapy to afford added protection for persons living in or traveling to areas where WNV is endemic. Although it would be desirable to obviate the biosafety level 3 requirements associated with WNV neutralization testing on a routine basis, cross-reactivity of ELISAs with other flaviviruses limits their utility for the intended purpose [27]. Also, there was no good correlation between the WNV neutralization titer of IVIG lots and the cumulative WNV incidence in the respective counties from where the plasma had been collected. As a consequence, WNV neutralization testing for individual IVIG lots will be required to identify high WNV antibody titers.

The successful treatment of established WNV infection in immunosuppressed patients by IVIG that contains WNV neutralizing antibodies has also been reported [11, 28]. Given that, at present, clinicians have few options other than supportive care for severe cases of WNV infection [29], the availability of a mechanistically targeted and well-understood medical intervention would be welcome and is under clinical evaluation [30]. With the large number of severe WNV cases that occur annually (i.e., 1433 neuroinvasive cases reported in 2006 alone [19]), an IVIG supply from a small donor base will likely be a limitation to treatment. As in the present study, IVIG lots from US plasma have been shown to be equally functional, so the use of products with high WNV neutralization titers for treatment as well as prophylaxis seems possible.

The correlation between WNV neutralization as measured by a functional assay in vitro and protection against an otherwise lethal WNV infection in vivo as demonstrated in our mouse experiments indicates that the in vitro assay should be useful in revealing an important functional parameter for IVIG lots-that is, WNV neutralization as a surrogate for protection against WNV infection. The neutralization titer of the product lot used for in vivo protection experiments is statistically equivalent to the WNV neutralization titer of the top 28% of all the lots analyzed (figure 1). As a consequence, all these product lots should be equally useful for passive protection applications.

Acknowledgments

We acknowledge Dr. Don Baker, for providing unconditional support and strategic vision for the project reported; Dr. P. Noel Barrett, for facilitating animal experiments and for long-term encouragement; Dr. Johann Kurz, for fruitful discussions and for mentoring a diploma thesis based on the work; the entire Pathogen Safety Team, most notably Bettina York, Claudia Schwarr, Karin Berka, and Elisabeth Pinter (for cell culture and virus propagation), Dr. Andreas Berting, and Dr. Gerhard Poelsler, for numerous discussions and scientific advice; Dr. Reinhard Ilk, for statistical support; Jean Marie Noel, for providing access to different lots of intravenous immunoglobulin; and Dr. Helga Savidis-Dacho, Elisabeth Hitter, Josef Mayrhofer, and Dr. Georg Holzer, for help with animal experiments.

Potential conflicts of interest: all authors are employed by Baxter BioScience, a manufacturer of plasma derivatives.

Financial support: Baxter BioScience.