Efficacy of rabies immunoglobulins in an experimental post-exposure prophylaxis rodent model

Vaccine 22 (2003) 244–249

Efficacy of rabies immunoglobulins in an experimental post-exposure prophylaxis rodent model Alexandre Servat a,∗ , Charles Lutsch b , Valentine Delore b , Jean Lang b , Keith Veitch b , Florence Cliquet a a Laboratoire d’études et de recherches sur la rage et la pathologie des animaux sauvages, Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Domaine de Pixérécourt, B.P. 9, Nancy, 54220 Malzéville, France b Aventis Pasteur SA, 2 Avenue Pont Pasteur, 69367 Lyon, France

Received 2 June 2003; accepted 20 July 2003

Abstract In a recently published Syrian hamster animal challenge study [Vaccine 19 (2001) 2273], a highly purified, heat-treated equine rabies immunoglobulin (pERIG HT, FavirabTM ) did not elicit satisfactory protection. The efficacies of this batch, a second stage pERIG HT batch and reference RIG preparations (ImorabTM , Imogam RageTM pasteurised, Berna antiserum) were compared in mice challenged with either Ariana canine field strain or CVS strain. Survival rates against Ariana challenge with the second pERIG HT batch were indistinguishable from those of other licensed preparations (83–90% survival), but the deficient batch did not provide satisfactory protection (53%). These data confirm the inadequate response to a first stage pERIG HT batch, but a current batch provides equivalent protection to that afforded by licensed HRIG and ERIG preparations. © 2003 Elsevier Ltd. All rights reserved. Keywords: Equine rabies immunoglobulin; Heat-treated; Post-exposure treatment

1. Introduction The gold standard for the early post-exposure prophylaxis (PEP) of patients exposed to possible rabies infection after bites from a suspected infected animal is based on the immediate local treatment of the wound—washing and disinfection, followed by local infiltration with anti-rabies immunoglobulins (RIG), and systemic treatment against the virus by anti-rabies vaccination at other body sites. When administered as soon as possible after the exposure, up to 7 days after exposure (time necessary to achieve the adequate virus neutralising antibody level following immunisation with a cell culture-derived rabies vaccine [2–4]) such prophylaxis has proven to be highly effective in avoiding mortality associated with untreated rabies infection. The low absorption rate and poor diffusion of these products, which elicit extremely low systemic antibody levels always below the required level [5–7], require careful and complete infiltration of the wound(s) rather than injection of the adequate dose of RIG intramuscularly at a distant site [4]. Although the immunological mechanism of action ∗

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by which immunoglobulins in wounds to neutralise rabies virus remains unclear, the local infiltration of virus inoculation sites has been shown to be a key element to confer protection in humans [6,7]. The efficiency and necessity of using equine immunoglobulins (ERIG) was soon established [8], in spite of the high incidence of allergic type reactions linked to the non-purified nature of the first sera used which contained a high proportion of heterologic proteins, including extremely immunogenic albumin. The original ERIG have been replaced in industrialised countries by human rabies immunoglobulins (HRIG) specifically developed during the 1970s [9] to avoid possible allergic reactions observed after treatment with early crude, heterologous preparations [10,11]. However, the high costs associated with the production of HRIG make the use of such preparations prohibitively expensive in those countries most affected by rabies in the developing world [12,13]. Purification steps introduced into the ERIG manufacturing process made it possible to eliminate the protein fraction responsible for most of the adverse effects, especially albumin and high molecular weight protein aggregates. Further additional steps to improve the safety profile included the use of pepsin digestion to split the Fc part of

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the immunoglobulin molecule, responsible for inflammatory reactions and complement activation, then removed by a precipitation step. This process yielded purified F(ab )2 fragments (the antigen binding fragment) containing the antigen binding site and the immune globulin recognition function. These most recent steps are necessary to abide by the stringent national, European and US regulatory requirements for the production of biological products. In the new ERIG preparation, heat-treated equine rabies immunoglobulin (pERIG HT, FavirabTM ), the proportion of F(ab )2 fragments containing the antigen binding site, is increased to 80–90% by three successive chromatographic steps, followed by a heat-treatment step to maximise viral safety [5]. Clinical studies in humans have shown that pERIG HT is equivalent to the previous purified (pERIG) preparation in terms of safety, immunogenicity, bioavailability and pharmacokinetics, and with a better clinical benefit-to-risk [5]. In a recent study, one batch of pERIG HT was shown to be poorly protective in an animal model (Syrian hamster) challenged with a virulent North American rabies virus strain [1]. The present report describes results of a study in a Swiss albino mouse animal model challenged with two different rabies strains. This confirms the poor efficiency of this batch when compared with an another batch of pERIG HT as well as the clinically proven RIG preparations. 2. Methods All studies were performed at AFSSA Laboratory, Nancy, France in collaboration with Aventis Pasteur Laboratories, Marcy l’Etoile, France, following guidelines of the European/French experimentation on animals.

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in the trigeminal muscle of the right paw in groups of 10 animals. Signs of rabies infection (health, illness, death) were monitored daily. Each manipulation was performed three times to determine the necessary dilution of the stock virus giving the mean LD95 in 50 ␮l. 2.3. Vaccine and Immunoglobulin preparations Aventis Pasteur supplied all immunoglobulin preparations and vaccine. Mice were vaccinated by administration of 50 ␮l (equivalent to one-tenth of the full dose) of VerorabTM (batch no. T0441-1, titre ≥2.5 IU by NIH, Aventis Pasteur) in the left trigeminal muscle. Five different immunoglobulin preparations were tested: • A first stage batch of pERIG HT (P6310) is an equine anti-rabies F(ab )2 fragment Heat Treated preparation from Aventis Pasteur. • A second stage batch of pERIG HT (T6195) is an equine anti-rabies F(ab )2 fragment heat treated preparation from Aventis Pasteur; the industrial process of production of this batch is strictly identical to the one used for the P6310 batch. The two batches P6310 and T6195 fulfilled the batch release specifications. • A HRIG HT preparation—Imogam RageTM Pasteurisée (Batch no. T0310) is a human anti-rabies IgG heat-treated preparation from Aventis Pasteur. • A pERIG preparation—ImorabTM (Batch no. R6486) is an equine anti-rabies F(ab )2 preparation from Aventis Pasteur. • An ERIG—Berna rabies antiserum (Batch no. 015 642 51) is an equine anti-rabies IgG preparation from Berna.

All experiments were performed using 21-day-old female Swiss albino mice (OF1 strain supplied by IFFA Credo, L’Arbresle, France).

The immunoglobulin preparation titre was measured using the mouse neutralisation test (MNT) and individual titres are shown in Table 1. All five preparations were diluted to allow administration of doses of 100, 200 and 400 IU/kg in a 50 ␮l injection. Each dilution was administered to a group of 10 animals.

2.2. Challenge virus strains

2.4. Study design

2.1. Animals

Two rabies virus strains were used for challenge: • Challenge virus strain 27 (CVS 27) is a standard, fixed laboratory strain produced in mice. • Ariana (reference 1009) is a canine field strain from North Africa (Tunisia), selected from a homogenate of salivary glands taken from a naturally infected dog by Haddad and Chappuis (1993, personal communication). This strain has been used recently to challenge dogs in an experiment of rabies DNA vaccination [14]. Aliquots of both strains were stored at −80 ◦ C until use. To determine the LD95 for each virus strain when administered by intramuscular injection, five 10-fold serial dilutions were prepared and 50 ␮l of each dilution was administered

Two series of experiments were performed, each with a total of 170 animals in 17 groups of 10 animals each. The Table 1 Rabies Neutralizing antibody titres of immunoglobulin preparations measured according to the mouse neutralisation test (MNT) expressed in international unit/ml Tested immunoglobulin preparations

Batch no.

pERIG HT

P6310 T6195

HRIG HT pERIG ERIG

T0310 R6486 015 642 51

a

Rabies antibody titre (IU/ml) 383 241 ≈200a 533 ≥200a

According to the specification of batch release. Not tested.

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Table 2 Protocol of virus, vaccine and immunoglobulin administrations in the 17 experimental groups (10 mice per group) Time

Group 1 (vaccine control)

Day −1

a

3 7 14 28

Groups 3–17

Survival rates results were compared for statistical significance using a χ2 -test at a 95% confidence level.

Viral challenge (CVS in series 1, Ariana in series 2)

Day 0 Day Day Day Day

Group 2 (virus control)

2.5. Statistical analysis

Vaccine Vaccine Vaccine Vaccine Vaccine

0.9% NaCl 0.9% 0.9% 0.9% 0.9% 0.9%

NaCl NaCl NaCl NaCl NaCl

Immunoglobulina Vaccine Vaccine Vaccine Vaccine Vaccine

100, 200 or 400 IU/kg of one each of the five preparations.

first set was performed with CVS as challenge strain and the second set with the Ariana strain. Animals were treated 24 h later. The chronology of the treatments is shown in Table 2. The first two groups were control groups, group 1 received virus and vaccine only, group 2 the challenge virus only. The remaining 15 groups received virus, vaccine and one of the three different doses of the five immunoglobulin preparations. Animals were then monitored daily for approximately 60 days for signs of rabies infection: health, illness and death. At the end of the surveillance period cardiac blood samples were taken from each of the surviving animals. After centrifugation at 5000 rpm for 15 min and incubation at 56 ◦ C for 30 min serum samples were stored at −20◦ C until the test of anti-rabies antibodies by the fluorescent antibody virus neutralisation (FAVN) test [15].

3. Results Survival rates for all the experimental groups at the end of the respective surveillance periods are shown in Table 3. Of the ten animals inoculated with CVS without any therapeutic treatment, only two survived. In the control group treated with Ariana three animals were lost due to a manipulation error, but all of the seven correctly inoculated mice died before the end of the experiment. These two survival rates (20 and 0%) were not significantly different (P = 0.05). Treatment with vaccine alone significantly increased the survival rates with both challenge strains (70 and 30% for CVS and Ariana, respectively). Comparing the two survival rates for animals treated with the same immunoglobulin and vaccine combinations, it is evident that rates were lower with the CVS strain than with Ariana. This was observed for all groups, irrespective of the immunoglobulin preparation, and the difference between the two strains was statistically significant for all immunoglobulin preparations. There was no dose-dependent effect with any of the immunoglobulins with either challenge strain; therefore the three dose groups with each preparation were pooled for further statistical analysis and presentation of the results.

Table 3 Survival rates of animals in all experimental groups with the two different challenge virus strains and five immunoglobulin preparations (10 animals per group per dose), and pooled data for each preparation (%) Immunoglobulin preparation

Dose (IU/kg)

Number of surviving mice from 10 inoculated (% survival rate of all doses − pooled data) CVS challenge

ARIANA challenge

HRIG HT

100 200 400

3 4 (37) 4

9 8 (83) 8

pERIG

100 200 400

1 1 (20) 4

8 9 (90) 10

ERIG

100 200 400

2 3 (20) 1

8 9 (83) 8

pERIG HT P6310

100 200 400

1 5 (27) 2

6 6 (53) 4

pERIG HT T6195

100 200 400

2 0 (20) 4

9 9 (87) 8

Vaccine alone CVS controls 10−2.8 ARIANA controls 10−1.5

7 (70) 2 (20) –

Values shown in the parentheses are in percentage. a Out of 10, only 7 animals correctly inoculated.

3 (30) – 0a (0)

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CVS 100 90 80

Survival rate (%)

70

Vaccine control CVS control pERIG HRIG HT ERIG pERIG HT P6310 pERIG HT T6195

60 50 40 30 20 10 0 0

5

10

15

20

25

30

35

40

Surveillance days

Fig. 1. Survival rates of mice challenged with CVS rabies virus strain then treated with rabies vaccine, with or without the respective immunoglobulin preparations. Each point represents the % of 10 (vaccine or virus control) or 30 animals (pooled data for three groups with different doses of immunoglobulin preparations) surviving at each time point.

Ariana strain 100 90 80 Vaccine control ARIANA control pERIG HRIG HT ERIG pERIG HT P6310 pERIG HT T6195

Survival rate (%)

70 60 50 40 30 20 10 0 0

5

10

15

20

25

30

35

Surveillance days

Fig. 2. Survival rates of mice challenged with Ariana rabies virus strain then treated with rabies vaccine, with or without the respective immunoglobulin preparations. Each point represents the % of 10 (vaccine or virus control) or 30 animals (pooled data for three groups with different doses of immunoglobulin preparations) surviving at each time point.

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3.1. Protection against challenge with CVS strain In animals challenged with CVS strain the survival rate varied between 0% (pERIG HT T6195) and 50% (pERIG HT P6310) in the individual groups. The best protection was conferred by Imogam RageTM (mean rate of 37%), but in all cases the rate was lower than that observed with vaccination alone (70%). There were no significant differences between the five groups (P = 0.05). 3.2. Protection against challenge with Ariana strain As already noted, survival rates with Ariana strain were higher than those with CVS, varying between 40 and 100% for individual groups. Moreover, no dose-dependent effects were observed. Mean rates (data for all doses pooled) varied from 83 to 90% for each of the immunoglobulin preparations with the exception of the early stage lot of pERIG HT (P6310). This preparation has the lowest mean survival rate (53%), which was significantly lower than the survival rates observed in the other groups (P = 0.05), and indeed was not significantly greater than vaccination alone (P = 0.05). The kinetic profiles of the survival rates are shown in Figs. 1 and 2 for the CVS and Ariana challenge strains, respectively. Fig. 1 clearly illustrates the poor protection afforded by all of the immunoglobulin preparations against the CVS strain. In comparison, protection against the Ariana strain was satisfactory with all preparations, with the exception of the P6310 lot of pERIG HT, which was slightly higher than the vaccinated-only group. 3.3. Sero-neutralising antibody titres in vaccinated mice Anti-rabies antibody titres in mice were determined by the FAVN test in the sera of surviving animals, but were not available from those mice that died during the surveillance period. Non-vaccinated animals had no detectable antibodies (titre <0.29 IU/ml) while a range of titres from 0.3 to 23.9 IU/ml was observed in individual surviving vaccinated animals in all groups. There was no evidence of any influence of the immunoglobulin preparations on these titres, with no differences in mean values between any groups including the vaccinated-only controls, and no apparent dose-dependent effect within any immunoglobulin-treated group. Furthermore, there were no differences in titres between animals challenged with CVS or Ariana virus strains.

4. Discussion The success of post-exposure prophylaxis in humans (wound washing, vaccination with or without RIG infiltra-

tion) is highly dependent on the punctuality of intervention, the severity and location of the wounds, the quality of the vaccine (cell culture), and the proper local instillation of RIG into all the wounds no later than 7–10 days after exposure, which is the time necessary to reach a protective neutralising antibody level. The physiopathological mechanism by which rabies virus reach the CNS is only partially understood. The humoral response provided by modern, potent cell-culture vaccines is known to be essential to stop a fatal progression of any infection. Indeed, an already immune patient does not need to receive local injection of RIGs. Several studies have shown that RIG alone, when administered at the recommended dose, are almost undetectable in the blood, and therefore cannot provide protection by themselves [16,17]. On the other hand, these products have proved their efficiency when administered in the site of virus entry in association with other rabies vaccine. This local neutralisation of the rabies virus is also essential in previously unimmunised subjects, but the extent of their action has not yet been determined. The present study was undertaken to investigate the efficiency of a first stage batch of pERIG HT batch (P6310) used by Hanlon et al. [1] in another animal model, with a more recent batch (T6195) and other established RIG preparations as comparators. Our results show that the protection conferred by the routine manufacturing batch (T6195) against challenge with either a laboratory standard strain (CVS) or a wild-type strain (Ariana) was indistinguishable from established preparations, but also confirmed the previous observation that the P6310 batch does not provide adequate protection against rabies. Despite the fact that the rabies antibody titre of the P6310 batch was in accordance with the specifications (383 IU/ml determined by the MNT), we may support the hypothesis of a lack of consistency of this first experimental preparation. Investigations are currently being conducted in order to further analyse these contradictory results of these preparations in post-exposure in vivo models. A second important point to note from our data is the difference in performance of all five RIG preparations against two different rabies virus challenge strains. None of the preparations provided adequate protection against challenge using the laboratory standard CVS strain. Indeed, were it not for the satisfactory performance of the established preparations against the Ariana strain, the results with the standard CVS would have raised major concerns over preparations which have otherwise been proven effective in clinical use. The reason for the discrepancy between the responses to the two strains may reflect the previously noted differences in pathogenicity of different CVS strains [18]. The CVS strain is generally pathogenic for mice. Among other factors, susceptibility of a host depends on the virus dose; therefore a better understanding of the pathogenesis of the Ariana strain in this post-exposure experiment in mice would require additional assays with different doses of virus. Fixed laboratory challenge strains are known to result in more abundant production of virus particles and more severe cytopathic

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changes than street strains of rabies virus [19]. The results obtained with the Ariana strain, which was isolated from an infected dog, may be considered to be more representative of the street strains expected to be encountered during clinical use of pERIG HT. In their model, Hanlon et al. [1] observed a survival rate of 100% in Syrian hamsters challenged with a canine rabies wild variant (from a Texan coyote) strain, and then treated with rabies vaccine together with HRIG HT or pERIG for a 75-day surveillance period. However, by day 20 the pre-release batches pERIG and pERIG HT P6310 had survival rates of only 67 and 22%, respectively. These lower survival rates with two early lots of pERIG and pERIG HT are partially confirmed by ours when considering the 50% survival rate with the early pERIG HT batch (P6310), but are in contradiction with the satisfactory results reported for the recent lot of pERIG HT (T6195) and the other products. When trying to reconcile these differences in terms of survival it is meaningless to presume the superiority of one model over the other. However, we must consider and reflect on which of the different parameters within the two models may have lead to these divergent results, i.e. the viral load used for challenge, the strain virulence as expressed by the measured LD50 , and the animal model used. More recent data published by Hanlon et al. [20] with a dog model provided similar figures with another experimental lot of pERIG HT [20]. Having all these parameters in parallel, it is difficult to assess their respective weight on the survival evaluated in these two studies using the same products. Furthermore, it should be noted that in vivo models using death of animals have demonstrated their limits for assays consisting in the titration of rabies neutralising antibodies by the MNT. Previous collaborative studies [21,22] conducted in different laboratories using the same protocol as well as the same animal model and biological products have shown a lack of reliability and reproducibility of results mainly due to uncontrollable factors such as susceptibility of the host to lethal infection. In conclusion, our data from a mouse model confirm the observation of Hanlon et al. [1], that the early batch of pERIG HT (P6310) does not provide adequate protection against rabies challenge. However, the more recent batch (T6195) provided protection that was indistinguishable from that conferred by licensed ERIG and HRIG preparations, which themselves have been proven efficient through extensive clinical use. It should be emphasised that although animal models are invaluable in testing such products, clinical studies on humans being ethically impossible, these studies cannot be extrapolated to the human situation. Therefore, the results of such pre-clinical rodent studies should always be treated with caution. Further studies performed through collaborations with different laboratories using different models are needed to confirm these results so as to reinforce the benefit-to-risk ratio of the pERIG HT preparation when it is administered in association with a cell-culture rabies vaccine.

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