Abstract

The present study sought to determine whether increasing and accelerating rabies vaccine administration would result in earlier protective levels of neutralizing antibody. Results indicated that the 8-site and double-dose Thai Red Cross intradermal regimens produced higher antibody titers by day 14 but not significantly higher titers by days 5 and 7. Administration of rabies immunoglobulin into and around bite wounds on the first day of rabies prophylaxis should remain the optimal postexposure treatment.

One of the last 2 international commercial manufacturers of equine rabies immunoglobulin discontinued production by 2001 and is now rapidly exhausting remaining supplies [1]. Human rabies immunoglobulin is in short supply. It is virtually unavailable or is not affordable in developing countries, which experience the majority of rabies exposures and human deaths [2–4]. The result is that >90% of patients who receive rabies postexposure prophylaxis worldwide are not given any human rabies immunoglobulin [4]. The introduction of highly immunogenic multisite intradermal (ID) rabies vaccine schedules >2 decades ago gave hope that one might be able to achieve earlier and higher neutralizing antibody (NAb) levels by use of one of these regimens. The 2 World Health Organization (WHO)-approved ID postexposure schedules are the Thai Red Cross (TRC-ID) and Oxford 8-site regimens [5–7].

In the absence of human rabies immunoglobulin, some rabies control centers have been applying the 8-site ID regimen, hoping that there might be an earlier immune response that could intercept the virus at the inoculation sites. However, there are no data on the failure of the infection to respond to treatment in such patients, and there are no controlled efficacy studies that allow us to call this an evidence-based practice. This topic was extensively discussed at 3 WHO-sponsored rabies conferences (17–20 July in Geneva, Switzerland; 20–21 December 2001 in New Delhi, India; and June 2002 in Manila, the Philippines) that considered the unavailability of rabies immunoglobulin in most of the world to be a true emergency. It was pointed out that old data, reviewed retrospectively, did not lend much hope for a significantly earlier immune response when rabies antigen delivery was increased by using multisite injections [1]. A prospective, controlled immunogenicity study was therefore recommended. Here, we report the results of such a newly completed study.

Methods. We recruited 89 volunteers with no previous history of rabies vaccination from Chulalongkorn and Kasetsart Universities and from the Queen Saovabha Memorial Institute. None of the volunteers had experienced a rabies exposure during the study period. Most volunteers were young, healthy veterinary students, nurses, or scientists who agreed to have blood samples drawn for determination of the presence of NAb. The locally obtained stock vaccine was manufactured by Aventis-Pasteur (Purified Verorab Rabies Vaccine, batch U-0160; potency at 5°C; 6.9 IU per lyophilized ampoule; supplied with 0.5 mL saline diluent). The study was approved by the ethics committees of Kasetsart University and the Queen Saovabha Memorial Institute and followed standards set in guidelines from the modified Helsinki Declaration.

Volunteers were randomly assigned to 4 different groups. Group 1 (n = 22; age 25 ± 11 years; 12 men and 10 women) received the 8-site ID regimen, normally consisting of 0.1 mL of vaccine injected at 8 sites on day 0, at 4 sites on day 7, and at 1 site on days 28 and 90 [6, 8, 9]. The series of vaccination in this group was shortened to injections on days 0 and 7 (8-0-4), omitting those normally given on days 28 and 90. Group 2 (n = 22; age 20 ± 2 years; 9 men and 12 women) received a double-dose TRC-ID regimen consisting of 0.1 mL of vaccine injected at 4 sites on days 0, 3, and 7 (4-4-4), omitting the injections on days 28 and 90. Group 3 (n = 22; age 21 ± 4 years; 5 men and 17 women) received the usual TRC-ID regimen consisting of one 0.1-mL ID injection at 2 sites on days 0, 3, and 7 (2-2-2), omitting the injections on days 28 and 90 [6, 10]. Group 4 (n = 23; age 21 ± 5 years; 10 men and 11 women) received the reference standard Essen postexposure schedule consisting of 1 intramuscular (IM) injection of 1.0 mL on days 0, 3, 7, and 14; the injection on day 28 was omitted [11].

All the volunteers have been scheduled for a booster vaccine injection and for determination of rabies-neutralizing antibody (RNAb) levels 1 year later, thus completing a reliable preexposure regimen in each group, suitable for individuals in occupations that are potentially exposed to rabies virus.

Serum was collected on days 0, 3, 5, 7, and 14 for the determination of RNAb levels at the immunology laboratory of the Queen Saovabha Memorial Institute via the rapid fluorescent focus inhibition test method, as described elsewhere [11]. The cutoff value, called the “detectable level” in this study, was 0.06 IU/mL.

Results. Data indicated that there were no detectable RNAbs in any of the groups on day 5, except for 2 patients in group 2 who had RNAb levels just above the cutoff point at 0.07 IU/mL. This is why we did not test for RNAbs on day 3 samples that had been stored for a future study. Seroconversions at day 7 were seen in 86% (19/22), 90% (19/21), 71% (15/21), and 29% (6/21) of patients in each of the 4 respective study groups. Geometric mean titers (GMTs) on day 7 were as follows: group 1, 0.12 IU/mL (range, <0.06–0.50 IU/mL); group 2, 0.14 IU/mL (range, <0.06–0.57 IU/mL); group 3, 0.08 IU/mL (range, <0.06–0.12 IU/mL); and group 4, 0.06 IU/mL (range, <0.06–0.1 IU/mL). All subjects in all groups had RNAb levels well above the WHO-recommended level of 0.5 IU/mL on day 14 (table 1). Data from study group 1 (8-site ID) indicated results similar to those for group 2 (double-dose TRC-ID). There was no statistically significant difference between RNAb levels in groups 1 and 2 on days 7 and 14 (table 1).

Table 1

Serologic response to multisite intradermal (ID) and intramuscular (IM) rabies vaccination in 4 study groups, according to vaccination schedule.

Table 1

Serologic response to multisite intradermal (ID) and intramuscular (IM) rabies vaccination in 4 study groups, according to vaccination schedule.

The GMTs in groups 3 (2-site ID) and 4 (IM) also showed similar results, and there was no significant difference in RNAb levels on days 7 and 14. The GMTs in the multisite ID groups (groups 1, 2, and 3) on day 7 were significantly higher than those in IM group 4 (P <.005). The GMTs in the 8-site and double-dose TRC-ID groups were significantly higher than those in the 2-site TRC-ID group and in the IM group on days 7 and 14 (P <.005). Adverse effects were mild and comparable with those seen in previous studies (table 2).

Table 2

Adverse reactions after intradermal (ID) and intramuscular (IM) rabies vaccination, by injection (inj).

Table 2

Adverse reactions after intradermal (ID) and intramuscular (IM) rabies vaccination, by injection (inj).

Discussion. It is generally accepted that vaccine alone, when any of the WHO-approved postexposure treatment regimens is used, will protect the majority of rabies-exposed patients and will do so without the use of immunoglobulin [2, 10]. A large viral inoculum, provided close to a peripheral nerve, followed by early entry of virus into the immune-protected neural environment, may doom a patient before endogenously formed humoral antibodies can intercept the virus [2, 8]. For this reason, the WHO Expert Committee on Rabies, the US Centers for Disease Control and Prevention, and the Thai Red Cross Rabies Committee recommend injecting the bite sites with immunoglobulin at the initiation of rabies postexposure prophylaxis [9, 10, 12].

The 2 ID schedules (used in groups 1 and 3), if properly applied, can save up to 70% in vaccine cost and result in equal or higher RNAb levels than expected with the reference standard IM Essen regimen [6]. This is a very important advantage in poor countries where rabies is endemic [3]. It has also been suggested that increasing the early vaccine antigen content, as is done with the 8-site ID or double-dose TRC-ID schedules, might induce an earlier immune response. It has been hypothesized that such regimens might be more efficient in neutralizing virus at bite sites before it invades the immune-protected neural environment [6, 7, 10].

There is a worldwide shortage of immunoglobulins. This has led to the recommendation to use the 8-site schedule if no immunoglobulin is available, because it is known to induce higher RNAb levels by day 14 [7, 10]. However, no scientific evidence to support this suggestion was ever offered. We found undetectable RNAb levels by day 5 in nearly all subjects. NAb titers barely approaching the 0.5 IU/mL level were seen in only 9%–10% of subjects by day 7. These data do not exclude some beneficial effects of the 8-site or double-dose TRC ID regimens over the IM (Essen) and conventional TRC-ID regimens. It is, however, unlikely that the use of these accelerated ID schedules will provide optimal postexposure therapy for all severely rabies-exposed patients. There was no significant difference in the immune response on days 7 and 14 between group 1 (8 site) and group 2 (4 site). The latter, however, proved to be more acceptable to the volunteers who avoided the added injections into the abdominal wall and intrascapular region required for the Oxford 8-site method.

Every effort needs to be made to bring back an adequate worldwide supply of affordable human and equine rabies immunoglobulins. There is a need to stimulate monoclonal antibody production, and new biologicals or antiviral drugs may someday be able to replace immunoglobulins of human or equine source.

Acknowledgments

We thank Charles E. Rupprecht, V. Sitprija, and T. Hemachudha, for critical comments.

Note Added in Proof

Note Added in Proof. A new chromatography purified and pasteurized equine rabies immunoglobulin made by Adventis-Pasteur in France is now being introduced in some countries where rabies is endemic.

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This research was carried out at the request of the Rabies Advocacy group of the World Health Organization. Rabies vaccines were purchased on tender from major international manufacturers.
Financial support: Thai Red Cross Society; Aventis-Pasteur, Thailand; Vijdusit Foundation.

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