Serum Neutralizing Antibody After Rabies Postexposure Prophylaxis LAWRENCE COREY, M.D.; MICHAEL A. W. HATTWICK, M.D.; GEORGE M. BAER, D.V.M.; and JEAN S. SMITH, B.S.; Atlanta, Georgia

One hundred seventy-seven persons submitted specimens for serum neutralizing antibody titer detreminations 30 to 90 days after starting postexposure rabies prophylaxis. Ninety-two percent of those who received duck embryo vaccine alone developed adequate antibody titers. However, 2 3 % of those who received equine antirabies serum plus duck embryo vaccine failed to develop an adequate antibody titer; one of these inadequate responders subsequently died of rabies. Factors that increased the risk of a poor antibody response included the receipt of steroids during the course of postexposure prophylaxis, and the use of more than 55 lu/kg of equine antirabies serum. Many persons receiving postexposure rabies prophylaxis fail to develop adequate humoral immunity and may have an increased risk of developing rabies. We suggest that persons receiving postexposure rabies prophylaxis should have serum neutralizing antibody determinations 3 0 to 4 0 days after starting treatment.

ALTHOUGH HUMAN RABIES is rare in the United States,

about 30 000 persons each year receive postexposure rabies prophylaxis. Since the introduction of this treatment by Pasteur in 1885, millions of persons have received courses of 12 to 28 or more doses of rabies vaccine. However, the efficacy of human postexposure rabies prophylaxis with rabies vaccine alone, especially in severe rabies exposures, has never been clearly shown (1 -3). Many comprehensive surveys have concluded that postexposure prophylaxis with vaccine alone is only partially successful in reducing the mortality from rabies, with efficacy levels similar to those achieved solely by local wound therapy such as cautery and cleansing with quanternary ammonium compounds (4-9). In the 1950s it was established that in both humans and animals, the early addition of passive humoral antibody to the vaccine regimen through the instillation of hyperimmune antirabies serum significantly increased the efficacy of postexposure rabies prophylaxis (10-14). Although the mechanism by which this regimen of postexposure prophylaxis protects has not been clearly established, neutralizing antibody, interferon, or both, appear to act while the virus is still at or near the site of inoculation (15-18). Thus, at • From the Bureau of Epidemiology, Center for Disease Control, Public Health Service, U.S. Department of Health, Education, and Welfare; Atlanta, Georgia.

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present, optimum postexposure rabies prophylaxis consists of local wound care, the administration of passive rabies antibody, and rabies vaccination (19, 2 0 ) . The addition of passive antirabies antibody was shown, however, to reduce the "active" humoral antibody response to most rabies vaccines. With nervous tissue rabies vaccine, additional "booster doses" of vaccine remedied this immunologic suppression (21-23). Rabies vaccine made from fixed virus grown in duck embryo and inactivated with beta-propriolactone was introduced in 1958 and, because of the lower risk of neurologic complications, has totally replaced nervous tissue vaccine for both preexposure and postexposure rabies prophylaxis in the United States ( 2 4 ) . Duck embryo vaccine, like nervous tissue vaccine, has been shown to have rather low immunogenicity, with multiple doses of vaccine needed to produce detectable levels of serum neutralizing antibody. Because of the relatively similar potency of duck embryo vaccine and nervous tissue vaccine, it was assumed that additional doses of duck embryo vaccine would also overcome the immunosuppressive effects of passive antibody (25-27). Two recent reports, however, suggest that some persons may not be developing high levels of serum neutralizing antibody after postexposure prophylaxis with equine antirabies serum and duck embryo vaccine (28, 29). In addition, rabies encephalitis that occurs despite postexposure prophylaxis with duck embryo vaccine plus equine antirabies serum has been associated with failure to develop high serum neutralizing antibodies after therapy (30-33). Our paper reviews, in persons epidemiologically exposed to rabies, the rabies antibody responses to the currently recommended regimens of postexposure rabies prophylaxis. Methods

As part of its nationwide surveillance of animal and human rabies, the Center for Disease Control since 1971 has asked attending physicians and state health departments to fill out questionnaires on all persons who have serum specimens tested for rabies neutralizing antibody. Our study analyzes data from those persons who had received postexposure rabies prophylaxis. Between June 1971 and March 1974, 651 persons who had received postexposure prophylaxis had serum neutralizing antibody titer determinations performed. Two hundred twentyone (34%) had received equine antirabies serum plus duck embryo vaccine, and 430 (66%) had received duck embryo vaccine alone. Eighty-eight (40%) of those who had received Annals of Internal Medicine 85:170-176, 1976

equine antirabies serum plus duck embryo vaccine, and 89 (21%) of those who had received duck embryo vaccine alone, had their antibody titers determined 30 to 90 days after starting therapy, the period when active neutralizing antibody to rabies is greatest. These 177 persons form the basis of our study. Demographic and epidemiologic data on the time and type of postexposure therapy given were available on all persons. Data on the animal involved in the exposure were available on 166 patients (94%). Ninety-eight persons (59%) were exposed to domestic animals: dogs, 28%; cats, 19%; cows, 9%; and horses, 2%. Sixty-eight persons (41%) were exposed to wild animals: bats, 13%; skunks, 10%; racoons, 1%; and foxes, 2%. Forty-five percent (75 persons) were exposed to laboratory-confirmed rabid animals: 51% of domestic animal exposures, and 37% of wild animal exposures. Information on the presence or absence of adverse reactions was available on 129 patients (73%)—70 recipients of duck embryo vaccine alone and 59 recipients of duck embryo vaccine and equine antirabies serum. Adverse reaction rates were calculated as the percent of persons on whom these data were available. Additional clinic, hospital, or physician records were obtained for patients who showed systemic reactions to equine antirabies serum plus duck embryo vaccine. Collection of Specimens and Laboratory Procedures

All serum specimens were shipped to the Viral Zoonoses Branch, Center for Disease Control, Lawrenceville, Georgia, where titers of neutralizing antibody to rabies were determined by the intracerebral mouse inoculation (MI) test or the rapid fluorescent focus inhibition (RFFIT) test. In the MI test done on all specimens submitted before 1 April 1973, dilutions of test serum were incubated with a challenge dose of live rabies virus and the mixture injected intracerebrally into mice. Serum specimens were screened at initial dilutions of 1:5 and 1:50. The serum neutralizing antibody titer of the test serum was the highest dilution that protected at least Vz of the injected mice, as calculated by the Reed-Muench technique (34, 35). Specimens submitted after 1 April 1973 were examined by the RFFIT test (36), which was done by mixing dilutions of test serum with a constant amount of tissue-cultureadapted rabies virus (CVS-11) (50 tissue-culture infecting doses/0.1 ml). The mixture was allowed to react in a C0 2 incubator for 90 minutes. Baby hamster kidney (BHK-21-13S) cells suspended in a diethylaminoethyl dextran-containing growth medium were added to the test chambers. The cultures were incubated in a C0 2 incubator at 35 °C for 24 h. The cultures were then washed, fixed, and stained with rabies fluorescent antibody conjugate and observed under a fluorescent antibody (FA) microscope. Twenty fields at magnification X 16 were read in each test chamber and were compared with virus controls. A reduction of 50% or more in the number of fields containing fluorescent cells indicated neutralizing antibody in the test serum. A neutralizing antibody titer of > 1:5 by the MI test was considered evidence of a definite antibody response to rabies vaccine. Titers > 1:50 were not end-pointed. To date, in our laboratory, both MI and RFFIT testing of serum samples has shown that a neutralizing antibody titer of > 1:16 by the RFFIT test likewise indicates the definite presence of active neutralizing antibody against rabies virus. Because the presence of humoral immunity as measured by an MI test titer of > 1:5 at the time of exposure to rabies has been associated in animals with protection against rabies virus (37, 38), titers of > 1:5 by the MI test or > 1:16 by the RFFIT test were considered adequate. Titers between 1:5 and 1:16 by the RFFIT test were considered low titers, while titers of < 1:5 by either test were considered negative. Because of occasional false-positives in low dilutions such as 1:8 and 1:12 with the RFFIT test and < 1:5 with the MI test, negative and low titers were considered inadequate; that is, they were not high enough to assure that an active antibody response to rabies had occurred.

Results SEROLOGIC RESPONSE

Table 1 indicates the overall serologic response to therapy. Fifteen percent of the 177 persons who received postexposure prophylaxis had inadequate serum neutralizing antibody titers at 30 to 90 days after starting therapy. Eight percent of the 89 persons who received 14 doses of duck embryo vaccine alone had inadequate serum neutralizing antibody titers, while 23% of those who received equine antirabies serum plus duck embryo vaccine had inadequate titers. All persons who received duck embryo vaccine alone, and all who received antirabies serum plus 21 or fewer doses of duck embryo vaccine, had their antibody level determined at least 9 days after their last dose of duck embryo vaccine. Of those who received antirabies serum plus all 23 doses of rabies vaccine, serum was drawn 1 to 7 days after the second booster in 44%, 8 to 14 days after the second booster in 17%, 15 to 21 days after the second booster in 7%, and 22 to 52 days after the second booster in 32%. The number of inadequate responders was two of 18, one of seven, two of three, and four of 16 in each group, respectively. No significant differences in the percentage of persons with inadequate antibody titers were noted between these groups; two (11%) of 18 persons who had antibody levels determined within 7 days after the second booster had inadequate titers, compared with seven (25%) of 28 persons who had their serum tested ^ 8 days after the second booster dose (P > 0.05). DUCK EMBRYO VACCINE RECIPIENTS

Eighty-two (92%) of 89 persons who received rabies vaccine alone had an adequate antibody response. Of the seven persons who had inadequate titers, two had received < 14 doses of duck embryo vaccine (11 and seven doses, respectively). Seventy-four percent of those receiving 14 doses of duck embryo vaccine had neutralizing antibodies of ^ 1:50; 18% had antibody titers between 1:5 and 1:50. All 20 persons who received more than 14 doses of duck embryo vaccine developed adequate neutralizing antibody activity; 19 of these persons had titers of ^ 1:50 (Table 2 ) .

Table 1. Neutralizing Antibody Titers at 30 to 90 Days in Recipients of Postexposure Rabies Prophylaxis Type of Treatment

Patients with Inadequate Serum Neutralizing Antibody Titer*

%

no.

20 (23)

68

(77)

88

7 (8) 27 (15)

82 (92) 150 (85)

89 177

%

•Mouse inoculation test titer< 1:5; rapid fluorescent focus inhibition test titer < 1:16. Corey et a/. • Postexposure Rabies Prophylaxis and Antibody

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Total Patients

no.

no, Equine antirabies serum plus duck embryo vaccine Duck embryo vaccine alone Total

Patients with Adequate Serum Neutralizing Antibody Titer

171

Table 2. Neutralizing Antibody in Recipients of Duck Embryo Vaccine Alone Doses Duck Embryo Vaccine Administered

no. 55 IU/kg of equine antirabies serum (P = 0.001). While the receipt of more than the recommended dose of 40 IU/kg of passive antibody was associated with a poor response to therapy, three of 14 persons who received the recommended dose of 40 IU/kg plus 23 doses of rabies vaccine still failed to respond adequately. Persons under 15 years of age were found to respond better to postexposure prophylaxis with antirabies serum plus duck embryo vaccine than those over 15. This response occurred despite the larger mean dose of equine antirabies serum given to this group, 77 IU/kg versus 47 IU/kg in adults. Since the dose of rabies vaccine administered to adults and children was the same (1 ml), this better response may be due to a greater antigenic mass per kilogram of body weight or to a better innate immune response to duck embryo vaccine by this age group. As noted in previous surveys (26), local reactions to duck embryo vaccine were common and were reported in 70% of our postexposure prophylaxis recipients. However, serious reactions such as anaphylaxis, hypotension, and bronchospasm were uncommon. One of 127 persons developed hypotension and cyanosis and had treatment discontinued. The side effects of generalized urticaria and serum sickness continue to be significant problems with 52% and 39%, respectively, of equine antirabies serum recipients developing these complications. The use of human rabies immune globulin will markedly reduce the morbidity of serum sickness from postexposure rabies prophylaxis, and for this reason human rabies immune globulin is preferable to equine antirabies serum (45-48). However, because human rabies immune globulin is more expensive than equine antirabies serum and may not always be available, its use when in short supply has been primarily recommended for persons who are allergic to equine serum, skin-test positive to antirabies serum, or pregnant

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(49). Thus, until human rabies immune globulin becomes universally available, equine serum will remain an important component of postexposure prophylaxis in this and other countries. Moreover, evidence indicates that the passive instillation of homologous antibody (human globulin) may suppress the active antibody response to duck embryo vaccine even more than heterologous (equine) antiserum (50). A controlled clinical study found that in young adult male volunteers, human rabies immune globulin and 14 doses of duck embryo vaccine failed to produce adequate serum neutralizing antibody titers in more than 40% of recipients. The percentage of recipients with inadequate titers was reduced to 8% when the full primary and booster series (23 doses of duck embryo vaccine) were administered (39). To test these observations under field conditions, the Center for Disease Control has initiated a prospective study of persons who receive postexposure prophylaxis with either form of hyperimmune rabies antiserum. In this series, one person with an inadequate titer after postexposure prophylaxis contracted rabies encephalitis and died. Available data suggest that patients who do not develop a good humoral antibody response to postexposure prophylaxis are at greater risk of developing clinical rabies than those who do. Review of the cases of rabies encephalitis in the United States since 1970 indicates that of the seven cases of rabies that have occurred, five received some form of postexposure prophylaxis, and three of these five were shown to have inadequate levels of neutralizing antibody against rabies after completion of their postexposure therapy. Of the two patients with adequate antibody titers, one recovered (30, 31, 51-55). Because as many as 20% of persons who receive the currently recommended dosage schedules for postexposure rabies prophylaxis may be at such risk, we recommend that each patient's antibody response be measured to evaluate its adequacy. In addition, since reaction rates and neutralizing antibody responses in persons who received their primary series over 14 days were the same or slightly better than in those who received their primary series over 21 days, we recommend that duck embryo vaccine be given as two doses a day for 7 days, followed by one dose a day for 7 days, so that those persons who do not respond adequately can be identified earlier. Patients at risk of developing rabies who have received postexposure prophylaxis with hyperimmune antirabies serum and vaccine should have their serum neutralizing antibody titer measured between 30 to 41 days after starting therapy, that is, 6 to 10 days after the first booster dose. Patients who have inadequate titers should have another antibody titer tested 7 to 10 days after the second booster dose of duck embryo vaccine. Antibody testing is especially important for those at high risk of having an inadequate antibody response, that is, persons over 15 years of age who received more than 55 IU/kg of equine antirabies serum; those who received steroids during the course of therapy; and those who received passive antibody but did not complete the full 21-dose primary series duck embryo vaccine.

Management of persons with inadequate titers must be done individually, and further prospective work on the response to additional booster doses of duck embryo vaccine or other rabies vaccines will be required before optimum management can be established (56-57). ACKNOWLEDGMENTS: Received 19 November 1975; revision accepted 19 April 1976. • Requests for reprints should be addressed to Lawrence Corey, M.D.; Viral Diseases Division, Center for Disease Control; Atlanta, GA 30245. References 1. VEERARAGHAVEN N, SUBRAHMANYAN T: Value of antirabies vaccine with and without serum against severe challenges. Bull WHO 22:381-391, 1960 2. WEBSTER LT: The immunizing potency of antirabies vaccines: a critical review. Am J Hyg 30:113-134, 1939 3. HILDRETH EA: Prevention of rabies. Or the decline of sirius. Ann Intern Med 58:883-896, 1963 4. MCKENDRICK AG: The ninth analytical review of reports from Pasteur Institutes on the results of antirabies treatment. Bull Health Organization, League of Nations 9:31-78, 1940 5. GREENWOOD M: Tenth report on data of antirabies treatment supplied by Pasteur Institute. Bull WHO 12:301-364, 1945 6. DEAN DJ, BAER GM, THOMPSON WR: Studies on the local treat-

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Serum neutralizing antibody after rabies postexposure prophylaxis.

Serum Neutralizing Antibody After Rabies Postexposure Prophylaxis LAWRENCE COREY, M.D.; MICHAEL A. W. HATTWICK, M.D.; GEORGE M. BAER, D.V.M.; and JEAN...
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