Rabies DNA vaccination of non-human primates: post-exposure studies using gene gun methodology that accelerates induction of neutralizing antibody and enhances neutralizing antibody titers

Vaccine 20 (2002) 2221–2228

Rabies DNA vaccination of non-human primates: post-exposure studies using gene gun methodology that accelerates induction of neutralizing antibody and enhances neutralizing antibody titers Donald L. Lodmell a,∗ , Michael J. Parnell a , John R. Bailey a , Larry C. Ewalt a , Cathleen A. Hanlon b a

b

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, 903 South Fourth Street, Hamilton, MT 59840, USA Rabies Section, Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, GA 30333, USA Received 6 November 2001; received in revised form 4 February 2002; accepted 6 February 2002

Abstract Pre-exposure DNA vaccination protects non-human primates against rabies virus. Post-exposure protection of monkeys against rabies virus by DNA vaccination has not been attempted. Presumably, post-exposure experiments have not been undertaken because neutralizing antibody is usually slow to be induced after DNA vaccination. In this study, we initially attempted to accelerate the induction of neutralizing antibody by varying the route and site of DNA vaccination and booster frequency. Gene gun (GG) vaccinations above axillary and inguinal lymph nodes or in ear pinnae generated higher levels of neutralizing antibody than intradermal (ID) needle vaccinations in the pinnae. Concurrent GG booster vaccinations above axillary and inguinal lymph nodes and in ear pinnae, 3 days after primary vaccination, accelerated detectable neutralizing antibody. GG booster vaccinations also resulted in higher neutralizing antibody levels and increased the durability of this response. Post-exposure vaccination with DNA or the human diploid cell vaccine (HDCV), in combination with an one-time treatment with human rabies immune globulin (HRIG), protected 50 and 75% of the monkeys, respectively, as compared to 75% mortality of the controls. These data will be useful for the refinement, development, and implementation of future pre- and post-exposure rabies DNA vaccination studies. Published by Elsevier Science Ltd. Keywords: Rabies virus; DNA vaccine; Booster; Neutralizing antibody; Post-exposure protection

1. Introduction More than 100 years ago, Louis Pasteur developed a crude desiccated nervous tissue vaccine for the treatment of rabies. The successful post-exposure treatment of 9-year-old Joseph Meister in 1885, with this nervous tissue vaccine initiated the era of human rabies prevention [1]. Unfortunately, in many developing countries, where rabies is endemic, individuals are still vaccinated with similar nervous tissue vaccines that are produced in brains of sheep, goats, or suckling mice, with ultraviolet or phenol inactivation of virus. Nervous tissue vaccines do not provide optimal protection and can cause adverse neurologic reactions. In contrast, vaccines produced in cell culture, such as the human diploid cell vaccine (HDCV) and the purified chick embryo cell vaccine, are very effective and well-tolerated, but they are prohibitively expensive for ∗

Corresponding author. Tel.: +1-406-363-9360; fax: +1-406-363-9380. E-mail address: [email protected] (D.L. Lodmell).

0264-410X/02/$ – see front matter. Published by Elsevier Science Ltd. PII: S 0 2 6 4 - 4 1 0 X ( 0 2 ) 0 0 1 4 3 - 3

people living in developing countries. It has been estimated that an Indian laborer would have to work 144 days to earn enough rupees to pay for the cost of post-exposure rabies prophylaxis for one patient [2]. Thus, improvements in rabies immunization may come from major advancements that have been made in the development of rabies DNA vaccines. Rabies DNA vaccines are very effective in the preexposure protection of mice, dogs and non-human primates [3–7]. Furthermore, DNA vaccination, in combination with an one-time dose of rabies immune serum, protects mice injected with rabies virus, 6 h previously [8]. Protection was associated with the elicitation of neutralizing antibody, as early as 5 days after the initial DNA vaccination. The accelerated induction of antibody resulted from multiple gene gun (GG) vaccinations in the skin above the axillary and inguinal lymph nodes, in combination with intradermal (ID) vaccinations in the ear pinnae, followed by booster vaccinations in similar sites. In this study, protocols were developed to accelerate the neutralizing antibody response

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of non-human primates after rabies DNA vaccination. Using these methods, we conducted a protection study of animals that had been injected with rabies virus, 6 h before vaccination with DNA or the HDCV, in combination with an one-time injection of human rabies immune globulin (HRIG).

2. Materials and methods 2.1. Plasmid construction Construction of the pCMV4 plasmid DNA, encoding the glycoprotein of the challenge virus standard strain of rabies virus, has been described [4].

Madison, WI) at 400 psi. The DNA was coated onto 0.5 mg of 2.1 ␮m-diameter gold beads according to the instructions provided by Powderject Vaccines. The ID DNA vaccinations (50 ␮g/0.1 ml) were given in the ear pinnae via needle. The HDCV (Pasteur Merieux Serums and Vaccins S.A., Lyon, France) was injected IM in the biceps femoris muscle. A heat-treated HRIG (Imogam Rabies HT, Pasteur Merieux Serums and Vaccines, S.A.) Lyon, France, distributed by Connaught Laboratories Inc., Swiftwater, PA, was injected (20 international units (IU)/kg) IM in the left and right masseter muscles. Blood was collected at pre-determined intervals from the cephalic vein, using a 20-gauge needle and a 5-ml syringe, or from the femoral triangle, using a 23-gauge needle and a 3-ml syringe. 2.4. Neutralizing antibody assay

2.2. Macaca fascicularis (Cynomolgus) The Macaca fascicularis(Cynomolgus) colony at the Rocky Mountain Laboratories (RML), Hamilton, MT, originated with captive monkeys from the Mauritius Islands. Eighteen animals (4–9 years) were wild-caught stock in the RML colony, while eight were captive-born in the RML colony. The monkeys (10 females, 16 males) were randomly assigned to experimental groups and ranged in age from 4 to 10 years. They were fed commercial high-protein monkey chow, supplemented with fresh fruit and commercial monkey treats. Automatic watering systems provided water ad libitum. None of the monkeys had been used in previous experimental research. Monkeys were routinely sedated with ketamine hydrochloride (10 mg/kg intramuscularly (IM)) for inoculations and blood sampling. The animal facilities, animal care and use programs at the RML are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, and they function in accordance with all United States Department of Agriculture, Department of Health and Human Services, and National Institutes of Health (NIH) regulations and standards. The monkeys were housed indoors in artificial light (12 h dark/12 h light cycle) alone, or in pairs, in aluminum barred cages (15.1 square feet floor space × 63 in. high or 6.3 square feet floor space × 32 in. high, respectively). The colony rooms are maintained at 21–26 ◦ C at 50% humidity. The 12 monkeys used in the viral challenge experiment were shipped to the Rabies Section, Centers for Disease Control and Prevention (CDC), Atlanta, GA by a domestic, commercial air freight provider. They were also handled in full accordance with all United States Department of Agriculture, Department of Health and Human Services and NIH regulations and standards. The husbandry of the CDC animal facility is similar to that of the RML facility. 2.3. Vaccinations, injections, and blood collections GG vaccinations at 2 ␮g/shot, were administered with the Dermal Powderject XR GG (Powderject Vaccines,

Rabies virus neutralizing antibody titers were determined as previously described [9,10]. Reciprocal dilutions of individual sera that reduced the number of fluorescent foci by 50% are shown in the figures. Antisera with known IU/ml of rabies virus neutralizing antibody, a rabies hyperimmune monkey serum, and the United States Standard Human Rabies Immunoglobulin R2 were included as positive controls in all neutralization assays. A titer of 1:20 was equivalent to 0.5 IU/ml, evidence of successful vaccination or immunologic priming. Normal monkey sera were included in the assays as negative controls. 2.5. Rabies virus challenge The monkeys were challenged by personnel in the Rabies Section, CDC. Virus was injected in the left and right masseter muscles with 0.5 ml of 1:5 dilution of a salivary gland homogenate obtained from a rabid dog, naturally infected with a coyote rabies virus variant [7]. The viral titer of the stock salivary gland homogenate was 106.5 mouse intracranial lethal dose50 /0.03 ml. After 6 h, the monkeys received a rabies DNA vaccine, control DNA vaccine, or HDCV. The first rabies DNA and HDCV vaccinations were accompanied with an one-time IM injection of 20 IU/kg HRIG infiltrated around the sites of virus injection. Booster vaccinations of DNA or HDCV, without HRIG, were given 3, 7, 14, and 28 days after viral challenge. The negative control monkeys used in this experiment also served as the negative controls in another non-human primate rabies DNA vaccine study, that was conducted concurrently with this study [11].The use of the same negative control animals in the two different experiments spared four control monkeys. After viral challenge, the monkeys were observed several times daily for clinical signs associated with rabies. At the first definitive clinical sign, animals were sedated and euthanatized with a barbiturate solution administered intravenously. At necropsy, brain impressions were made and tested for rabies virus antigen by the direct fluorescent antibody test.

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3. Results 3.1. Neutralizing antibody responses after different routes and methods of vaccination Successful post-exposure protection against rabies virus is most likely dependent upon the elicitation of a rapid protective neutralizing antibody response [12]. Thus, we initially determined whether different routes and methods of DNA vaccination could accelerate the elicitation of antibody in non-human primates. Control monkeys were vaccinated IM with HDCV. Neutralizing antibody was not detected in DNA- or HDCV-vaccinated monkeys on day 3 (Fig. 1). By day 7, both HDCV-vaccinated animals and an animal vaccinated with the GG above the lymph nodes were positive for antibody. After 3 days (day 10), titers of the antibody-positive animals had increased, and antibody was initially detected in a monkey who had been vaccinated via GG in the pinnae. Antibody levels increased in all antibody-positive animals at day 14, and five of the six

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monkeys on day 21, but remained undetectable until day 21 in monkeys who had been vaccinated ID via needle in the pinnae (Fig. 1). At this time, antibody titers in the other three groups were comparable averaging 1:960–1:1920. Thus, antibody was detected at least 14 days earlier in animals vaccinated via GG above the axillary and inguinal lymph nodes or in the ear pinnae than it was in animals vaccinated ID via needle in the pinnae. The ID needle vaccination in the pinnae also elicited the lowest and least durable antibody titers. Blood samples obtained 420–700 days after vaccination indicated that similar durable high levels of neutralizing antibody were maintained in monkeys that had received an IM vaccination of HDCV or DNA administered via GG (Fig. 1). 3.2. Evaluation of neutralizing antibody responses after multiple gene gun booster DNA vaccinations The previous experiment determined that DNA vaccinations with the GG elicited neutralizing antibody more

Fig. 1. Neutralizing antibody responses as determined using different methods or routes of DNA vaccination. Two monkeys each were vaccinated IM in each biceps femoris muscle with 0.5 ml of HDCV (䉱); ID via needle with 50 ␮g of DNA in each ear pinna (䊊); via gene gun (GG) with 20 ␮g of DNA in the pinna of each ear (five shots of 2 ␮g each) (䊉); or via GG with 40 ␮g of DNA above the axillary area of each arm and above the inguinal area of each upper thigh (five shots of 2 ␮g above each area) (䊏). At the designated intervals, blood samples were tested for neutralizing antibody. Six and three monkeys were available for blood sampling on days 420 and 700, respectively,. The horizontal dashed line (1:20, titer) indicates 0.5 IU/ml of neutralizing antibody, evidence of successful vaccination or immunologic priming. The numbers 1 and 2 above the symbols designate individual animals in each group.

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Fig. 2. Effect of multiple gene gun booster DNA vaccinations upon acceleration and augmentation of the neutralizing antibody response. Four monkeys (each designated by a different symbol) were vaccinated via gene gun above the axillary area of each arm, the inguinal area of each upper thigh and in each ear pinna with a total of 60 ␮g of DNA. Booster vaccinations identical to the primary vaccination were administered in identical areas 3, 7, 10, and 14 days after the initial vaccination. Blood samples were obtained from the animals prior to each booster and 21, 308, and 588 days after the initial vaccination. The sera were tested for neutralizing antibody. Three monkeys and one monkey were available for blood sampling on days 308 and 588, respectively. The horizontal dashed line (1:20, titer) indicates 0.5 IU/ml of neutralizing antibody, evidence of successful vaccination or immunologic priming.

quickly than did an ID needle vaccination. Next, we questioned whether primary and booster vaccinations with the GG in the pinnae and above the lymph nodes would further expedite the appearance of antibody. None of the monkeys was positive for neutralizing antibody 3 days after vaccination (Fig. 2). However, all monkeys had seroconverted 4 days after the first booster (7 days after primary vaccination). Levels of antibody at this time ranged 1:40–1:160, the titer of each monkey exceeding the antibody level of the only animal that had responded 7 days after an one-time GG vaccination (titer, 1:20) (Fig. 1). Ten days after primary vaccination (3 days after the second booster), antibody titers had increased from 16- to 64-fold (1:1280–1:2560) (Fig. 2), as compared with antibody levels that remained undetectable or much lower in animals who had received a single vaccination only (Fig. 1). Antibody levels continued

to increase after each booster vaccination, attaining an average titer of 1:81920, 21 days after primary vaccination (Fig. 2). At this time, the antibody level in monkeys receiving only a primary vaccination were 64-fold less, averaging just 1:1280 (Fig. 1). Thus, an antibody titer of at least 0.5 IU/ml (1:20) was present at 7 days in all animals receiving a booster, 3 days after primary vaccination. In addition to accelerating and enhancing antibody titers, the multiple boosters also resulted in the maintenance (308–588 days) of higher levels of antibody, as compared to animals who received a single vaccination (Figs. 1 and 2). 3.3. Post-exposure rabies DNA vaccination The successful acceleration of the induction of neutralizing antibody suggested that the DNA vaccine might

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be successful in protecting rabies virus-exposed monkeys. Hence, twelve animals (four per group) were challenged with the rabies virus. Six hours later, the monkeys received a rabies DNA vaccine, control DNA vaccine, or HDCV. The initial rabies DNA and HDCV vaccinations were accompanied with an one-time IM injection of HRIG (see Section 2). Three days after the viral challenge, low levels of neutralizing antibody were present in two of the four HDCV-vaccinated monkeys, but the other animals were negative for antibody (Fig. 3). Blood from two monkeys was not sampled at this time. By day 7, all (four out of four) HDCV-vaccinated monkeys had developed substantial levels of antibody, whereas lower antibody titers were

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detected in two DNA-vaccinated animals and two control animals. Between days 7 and 14, three negative controls, two DNA-vaccinated monkeys and one HDCV-vaccinated monkey developed rabies, and were euthanatized (Fig. 3). All monkeys developing rabies had neutralizing antibody titers >0.5 IU/ml (1:20). Among these animals, the HDCV-vaccinated monkey presented with the highest antibody titer, 7 days after virus challenge and at the time of euthanasia. Three monkeys vaccinated with HDCV, two monkeys vaccinated with DNA and one control animal remained rabies-free at 6 months. The antibody titers of the vaccinated survivors were similar, ranging from 1:320 to 1:960.

Fig. 3. Protection after post-exposure DNA vaccination. Twelve monkeys (three groups of four animals each) were challenged with rabies virus in each masseter muscle. Six hours after viral challenge, one group of monkeys received gene gun-administered rabies DNA vaccine in each ear pinna and above the axillary and inguinal lymph nodes (60 ␮g DNA total) and 20 IU/kg of heat-treated HRIG infiltrated around the virus injection site (䊏). A second group of monkeys received 1 ml of HDCV IM in the biceps femoris muscle and heat-treated HRIG infiltrated around the viral injection site (䊉). A third group of animals received gene gun-administered plasmid DNA (60 ␮g) without the inserted rabies virus glycoprotein gene ( ) at times and in anatomical areas identical to those for the group receiving the rabies DNA vaccine. Booster vaccinations, without HRIG, identical to the primary vaccinations were given 3, 7, 14, and 28 days after the primary vaccination. Blood samples were obtained at the designated times and tested for neutralizing antibody. The dashed horizontal line (1:20 titer) indicates 0.5 IU/ml of neutralizing antibody, evidence of successful vaccination or immunologic priming. The numbers 1–4 above the symbols identify individual animals in each group. Numbers within parentheses indicate the day an animal developed rabies and was euthanatized. The antibody titer corresponds to the blood sample obtained at the time of euthanasia.

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4. Discussion Successful post-exposure prophylaxis against severe rabies exposure traditionally consists of immediate passive protection with human or equine rabies immune globulin (RIG) inoculated at the site of virus exposure, and a series of rabies vaccinations usually administered IM on days 0, 3, 7, 14, and 28 to induce active immunity as the RIG levels decline [13]. In general, the brisk induction of high levels of neutralizing antibody correlates with protection. However, the mere induction of high levels neutralizing antibody or the absence of neutralizing antibody may not directly correlate with protection. In addition to rabies virus neutralizing antibody, non-neutralizing antibody to internal vital proteins [14,15], interferon [16], and possibly cell-mediated immunity [17,18] may play important roles in protection. The data presented here, clearly demonstrate that GG vaccinations above the axillary and inguinal lymph nodes, or in the ear pinnae accelerated the induction of neutralizing antibody. GG vaccinations also induced higher and more durable levels of antibody than did ID needle vaccinations in the ear pinnae. Furthermore, concurrent booster DNA vaccinations given above the axillary and inguinal lymph nodes and in the ear pinnae via GG accelerated and elevated antibody titers compared with those in animals who received only a GG primary vaccination in similar sites. Vaccinations administered ID in the ear pinnae were tested because similar vaccinations in mice evoke rapid and marked neutralizing antibody responses [8]. Vaccinations were not given IM or ID above the axillary and the inguinal lymph nodes because these routes/methods of rabies DNA vaccination have been shown to elicit either a slowly rising or minimal antibody response in monkeys [7]. Rabies booster DNA immunizations are usually given after an extended rest period between vaccinations [5–7,19–21] . Here, we showed that a lengthy rest period was not necessary to accelerate and augment the neutralizing antibody response. Neutralizing antibody levels indicative of successful vaccination were detected 7 days after a primary and one booster vaccination. The accelerated antibody response is more than likely to have occurred because the DNA was administered to multiple anatomical sites in the skin of the pinnae and abdomen that are adjacent to draining lymph nodes. The skin-associated lymphoid tissues contain specialized cells, such as keratinocytes, macrophages, and dendritic cells (e.g. Langerhans cells (LC)), all of which are involved in the initiation and augmentation of immune responses [22,23]. Furthermore, it has been shown that the skin and ear pinnae are excellent areas for initiating antibody responses with DNA vaccines [22–26]. It is also known that directly transfected LC are triggered to migrate from the skin to draining lymph nodes after entrance of GG propulsed plasmid-coated gold beads [26], and that DNA-expressing LC are present within draining lymph nodes within 24 h of GG vaccination [26].

The presence of neutralizing antibody, 7 days after a primary and one booster vaccination suggested that the DNA vaccine might be successful in protecting rabies virus-exposed non-human primates. However, the survival of an unvaccinated control monkey and the death of a positive control monkey who had received HDCV and HRIG, complicated interpretation of the protection study. There are numerous reports of rabies virus-exposed animals resisting infection or even recovering from infection [27–33]. Fekadu [34] determined that the number of dogs recovering from experimental rabies infection may vary from 0 to 20%, and that resistance/recovery is independent of either virus strain or virus dosage. To date, a definitive reason(s) for this resistance has not been determined. The survival of the unvaccinated control monkey in our study also raises questions concerning possible susceptibility differences of out-bred monkeys against rabies virus. On occasion, despite post-exposure administration of tissue culture vaccines and RIG, rabies post-exposure vaccine prophylaxis of humans fails to prevent rabies. Several suggestions have been proffered to explain the vaccine failures. These include inadequate wound care, delay in the initiation of treatment, failure to administer immunoglobulin with the vaccine, late administration of immunoglobulin, injection of an inadequate dose of hyperimmune serum, failure to infiltrate the bite site with immune globulin, and injection of hyperimmune serum into gluteal fatty tissue [35–38]. Underlying diseases, such as hepatitis or diabetes, and ongoing treatment with drugs that depress the immune system are also known to reduce the effectiveness of rabies prophylaxis [39–41]. Several of the possible reasons to explain rabies vaccine failure could apply to our study. We did not scrub the virus injection site (no local wound treatment), and we did not begin vaccination or treatment with HRIG until 6 h after viral challenge. Furthermore, due to concerns over potential adventitious agents, the Food and Drug Administration is recommending that immune globulin products be heat-inactivated. The HRIG product used in this study is heat-inactivated at 60 ◦ C for 10 h. Thus, in an effort to maximize safety, there might be a deleterious effect on the neutralizing potency of the HRIG, resulting in decreased efficacy. Although our in vitro testing indicated that the heat-inactivation did not compromise the potency of the HRIG, its’ efficacy in an in vivo model is not routinely tested. Furthermore, it recently has been shown that heat-treatment of equine RIG compromises its efficacy in a hamster model of rabies post-exposure prophylaxis [42]. The control monkeys did not receive HRIG because antibody alone does not protect non-human primates against rabies [42]. It may protect a few animals in a minority of cases but, usually it just prolongs the incubation period. After the protective activity of the HRIG decays, the animal remains immunologically-naive to rabies and susceptible to infection. Thus, to include an additional group of controls designated to receive HRIG, would only be redundant to the untreated controls.

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Injection of high concentrations of challenge virus in close proximity to the brain might also be considered as an explanation for the development of rabies in the HDCV- and DNA-vaccinated monkeys. A robust challenge was required because non-human primates are highly resistant to rabies virus [16,43,44]. Thus, in vaccination studies, a severe viral challenge dose must be used to ensure that at least 80% of the non-vaccinated controls die. Hence, rapid transit of virus from the masseter muscles to the central nervous system might have occurred before the HRIG or vaccine-induced neutralizing antibody could neutralize the virus. As a consequence of these potent viral challenges, superior vaccination treatments are needed to protect monkeys against rabies virus. Although the number of survivors in the DNA- and HDCV-vaccinated groups was not statistically different from the number of unvaccinated control monkeys that survived, the 50% survival of the DNA-vaccinated monkeys suggested that a DNA vaccine might eventually be used as a substitute for cell culture vaccines in post-exposure vaccination of humans. Repeating the experiment with larger sample sizes, and perhaps a different source of unheated HRIG, might result in statistically significant protection. The number of survivors not withstanding, we have clearly shown that rabies DNA GG vaccinations in the ear pinnae and above the axillary and inguinal lymph nodes elicited higher titers of neutralizing antibody than did ID needle vaccinations in the pinnae. In addition, concurrent GG booster vaccinations in the pinnae and above the lymph nodes of the arms and thighs accelerated the induction of neutralizing antibody. Antibody titers of monkeys receiving boosters were also augmented and more durable than levels elicited by a primary vaccination only. Lastly, similar durable (420–700 days) neutralizing antibody titers were maintained in animals who had been vaccinated with DNA or HDCV. These data should be useful for the refinement, development, and implementation of future pre- and post-exposure rabies DNA vaccination studies.

Acknowledgements We thank K. Hasenkrug, J. Portis, and S. Priola for critical review of the manuscript; W. Sheets, D. Dale, and the staff of the Veterinary Branch of the Rocky Mountain Laboratories; the Animal Resources Branch, Centers for Disease Control and Prevention, for care of the monkeys; G. Hettrick and A. Mora for graphic arts assistance; Nancy B. Ray for construction of the DNA vaccine; and C. Rupprecht for providing the challenge virus, and arranging for facilities to hold the rabies virus injected monkeys. We also thank many of the professional staff of the CDC Viral and Rickettsial Zoonoses Branch and the Animal Resources Branch, without whom this study would not have been possible.

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