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One-time gene gun or intramuscular rabies DNA vaccination of non-human primates: comparison of neutralizing antibody responses and protection against rabies virus 1 year after vaccination
Vaccine 20 (2002) 838–844
One-time gene gun or intramuscular rabies DNA vaccination of non-human primates: comparison of neutralizing antibody responses and protection against rabies virus 1 year after vaccination Donald L. Lodmell a,∗ , Michael J. Parnell a , John R. Bailey a , Larry C. Ewalt a , Cathleen A. Hanlon b a
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840, USA b 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, GA 30333, USA Received 25 May 2001; received in revised form 28 August 2001; accepted 29 August 2001
Abstract We have previously shown that Macaca fascicularis (Cynomologus) monkeys receiving a primary and either one or two booster rabies DNA vaccinations are protected against rabies virus. In this study, we determined whether monkeys that had been vaccinated only once via gene gun or intramuscularly (i.m.) with different concentrations of DNA would be protected against rabies virus challenge. Neutralizing antibody responses were assayed for 1 year before the monkeys were challenged. Neutralizing antibody was detected at least 50 days earlier in gene gun vaccinated as compared to i.m. vaccinated animals. Prior to viral challenge, all (6/6, 100%) gene gun vaccinated animals, but only 3/6 (50%) i.m. vaccinated animals seroconverted. In general, antibody titers of the gene gun vaccinated animals were higher than the titers of the i.m. vaccinated animals. There was no correlation between the concentration of DNA used for vaccination, the neutralizing antibody responses elicited and protection against viral challenge. Seven days after viral challenge, a rapid and strong anamnestic antibody response was elicited in 100% of the gene gun vaccinated monkeys and in four i.m. vaccinated monkeys. Neutralizing antibody remained undetectable in two i.m. vaccinated monkeys. Overall, 60% (3/5) of the gene gun vaccinated animals and 87% (5/6) of the i.m. vaccinated monkeys survived viral challenge. This study is the first, to our knowledge, to show long-term protection of non-human primates against a human viral pathogen using a DNA vaccination protocol that did not include a booster immunization. Published by Elsevier Science Ltd. Keywords: Rabies virus; DNA vaccine; Protection; Neutralizing antibody
1. Introduction Rabies, ‘la rage’ or madness, has been the object of human fascination, torment and fear since the disease was recognized in antiquity [1]. The World Health Organization (WHO) estimates that 40,000–100,000 human deaths are caused by rabies each year [2], and that 10–12 million people in developing countries receive one or more doses of rabies vaccine annually after exposure to the virus [3]. Many developing countries continue to use vaccines produced in sheep, goat or suckling mouse brain, with subsequent inactivation of virus with ultraviolet light or phenol [4,5]. The WHO recommends that vaccines derived from brain tissue be discontinued and replaced with vaccines produced in tissue culture cells [6]. Although vaccines derived from cultured cells such as the human diploid cell vaccine (HDCV) ∗ Corresponding author. Tel.: +1-406-363-9360; fax: +1-406-363-9380. E-mail address: [email protected] (D.L. Lodmell).
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are efficacious and well tolerated, they are costly to produce and prohibitively expensive to purchase. DNA vaccines offer a new and powerful approach for the generation of rabies vaccines. The advantages of DNA vaccines are their ease of construction, high temperature stability, cost effectiveness and ability to induce a full-spectrum of humoral and cellular immune responses [7]. Recently, we showed that Macaca fascicularis (Cynomolgus) monkeys given a primary and either one or two booster vaccinations of a rabies DNA vaccine encoding the glycoprotein of the challenge virus standard strain are 100% protected against rabies virus challenge [8]. The monkeys had been vaccinated via gene gun, or with a combination of intradermal and intramuscular (i.m.) injections. In addition, antibody elicited by the DNA-vaccinated monkeys neutralized in vitro a worldwide spectrum of rabies virus variants, suggesting that a single DNA vaccine could be used globally against the classical rabies viruses [8].
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2. Materials and methods
with a total of 8, 40, or 60 g of DNA. DNA was coated onto 0.5 mg of gold beads 2.1 m in diameter, according to instructions provided by PowderJect Vaccines, Madison, WI, and administered with the Dermal PowderJect XR gene gun at 400 psi. The DNA was administered at 2 g per shot. A booster vaccination was not given. Blood was collected at regular intervals and sera were tested for rabies virus neutralizing antibody.
2.1. Plasmid construction
2.4. Neutralizing antibody assay
Construction of the pCMV4 plasmid DNA vaccine encoding the G of the challenge virus standard strain of rabies virus has been described [9,10].
Rabies virus neutralizing antibody titers were determined as previously described [11,12]. Reciprocal dilutions of individual sera that reduced the number of fluorescent foci 50% are shown in the figures. Antisera with known International Units (IU)/ml of rabies virus neutralizing antibody, a rabies hyperimmune mouse serum and the United States Standard Human Rabies Immunoglobulin R2, were included as positive controls in all 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.
In this study, we (1) compared for over 1 year, neutralizing antibody responses of monkeys that had been vaccinated one-time i.m., or via gene gun, with different concentrations of DNA and (2) determined whether the monkeys were resistant to rabies virus challenge 1 year after vaccination.
2.2. M. fascicularis (Cynomolgus) The M. fascicularis (Cynomolgus) colony at the Rocky Mountain Laboratories (RML) originated with captive monkeys from the Mauritius Islands. Fourteen animals were wild-caught stock (4–9 years in the RML colony), while two were captive-born at RML. The monkeys (12 female, 4 male), randomly assigned to experimental groups, ranged in age from 4–10 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 hydochloride (10 mg/kg i.m.) for inoculations and blood sampling. The animal facilities and animal care and use programs at the RML are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Both function in accordance with all United States Department of Agriculture, Department of Health and Human Services, and National Institutes of Health Regulations and Standards. The monkeys were housed indoors in artificial light (12/12 h dark/light cycle) and housed paired or singly in aluminum barred cages (15.1 ft2 floor space×63 in. high or 6.3 ft2 floor space × 32 in. high, respectively). The colony rooms are maintained at 21–26◦ C at 50% humidity. Prior to virus challenge, the monkeys were shipped to the Rabies Section, Centers for Disease Control (CDC), Atlanta, Georgia by a domestic commercial air freight provider, in full accordance with all United States Department of Agriculture, Department of Health and Human Services and National Institutes of Health Regulations and Standards. The husbandry of CDC animal facility is similar to that of the RML facility. 2.3. Vaccinated groups of monkeys Twelve monkeys (two animals per group) were vaccinated i.m. in the triceps muscle with either 100, 500, or 1000 g of DNA, or via gene gun above the axillary area of each upper arm and above the inguinal area of each upper thigh
2.5. Rabies virus challenge The monkeys were challenged 375 days after vaccination at the Rabies Section Unit, CDC, Atlanta, GA. For viral challenge, 0.5 ml of a 1:5 dilution of a salivary gland homogenate obtained from a rabid dog naturally infected with a coyote rabies virus variant [8,13] was injected in the left and right masseter muscles. The viral titer of the stock salivary gland homogenate was 106.5 mouse intracranial lethal dose50 /0.03 ml. Previously, challenge in the masseter muscles with the same concentration of the identical virus preparation caused rabies in 100% of the negative control monkeys [8]. After challenge, the monkeys were observed several times daily for clinical signs associated with rabies. At the first definitive clinical sign, animals were sedated and euthanized with a barbiturate solution administered intravenously. At necropsy, brain impressions were made and tested for rabies virus antigen by the direct fluorescent antibody test.
3. Results The i.m. and gene gun routes of vaccination elicited completely different neutralizing antibody responses (Fig. 1A and B). As early as 7 days after vaccination, 4/6 gene gun vaccinated animals were positive for antibody, and at 60 days 6/6 animals were positive. In contrast, neutralizing antibody was initially detected in two i.m. vaccinated animals 60 days after vaccination. A third i.m. vaccinated animal seroconverted at 180 days, but three i.m. vaccinated monkeys remained antibody negative. Furthermore, antibody levels of the two groups were notably different. The titers of the i.m. vaccinated animals never exceeded 1:80, whereas gene
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gun vaccinated animals attained levels as high as 1:1280. The elicitation of antibody in i.m. vaccinated animals also appeared to be DNA dose-dependent. Antibody was not detected in monkeys vaccinated with 100 g of DNA, whereas 3/4 animals vaccinated with 500 or 1000 g of DNA seroconverted. A dose-dependent response was not evident among the gene gun vaccinated animals. At the time of viral challenge, neutralizing antibody was present in 4/6 gene gun vaccinated and 3/6 i.m. vaccinated animals (Fig. 1A and B). Three days after viral challenge, one gene gun vaccinated rabies negative monkey died of age-related causes. Neutralizing antibody responses of the remaining animals were notably varied (Fig. 1A and B) (five animals were not
bled on day 3). Titers had increased in 2/2 gene gun and 2/2 i.m. vaccinated animals, but remained negative in 2/2 i.m. vaccinated monkeys. Seven days after challenge, all gene gun vaccinated animals had high (1:240) to very high (1:122,880) levels of neutralizing antibody. Among the i.m. vaccinated monkeys at this time, antibody was initially detected in one animal, and the levels of neutralizing antibody of the three previously positive animals had increased significantly (128–2048-fold). In addition, 2/4 negative controls developed antibody by day 7, and all (4/4) were seropositive by day 14, or the day of euthanasia. Three negative control animals developed rabies 12 and 13 days after viral challenge. The fourth negative control
Fig. 1. Neutralizing antibody responses and protection of monkeys challenged with rabies virus 1 year after DNA vaccination. (A) Intramuscular vaccination. Two monkeys were vaccinated i.m. in the triceps muscle with 100, 500 or 1000 g of DNA. (B) Gene gun vaccination. Two monkeys were vaccinated via gene gun with 8, 40, or 60 g of DNA above the axillary area of each arm and above the inguinal area of each thigh (2 g per shot). Three hundred seventy-five days after vaccination the monkeys were challenged i.m. in the masseter muscles with rabies virus. The animals were anesthetized with ketamine hyrochloride and bled at the designated intervals, and the sera tested for neutralizing antibody. The horizontal dashed line (1:20 titer) in both panels A and B indicates 0.5 IU/ml of neutralizing antibody, evidence of successful vaccination or immunologic priming. The arrow (↑) designates the day of rabies virus challenge. The numbers 1–4 above the non-vaccinated control animals (∗) identify each individual animal in this group. Data from the same control monkeys are shown in panels A and B. The day monkeys developed rabies and were euthanized is shown in parentheses. The antibody titer corresponds with the terminal bleed. Three days after viral challenge, five monkeys were not bled, and one gene gun vaccinated rabies negative monkey died of age-related causes.
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Fig. 1. (Continued).
remained clinically normal and seropositive up to 210 days after challenge, but was rabies negative at euthanasia. Three vaccinated animals also developed rabies and were euthanized on days 12 and 13. The animal vaccinated i.m. with 500 g of DNA did not seroconvert after vaccination, and Table 1 Summary of seroconversions and protection of non-human primates vaccinated one-time with a rabies DNA vaccine, and challenged 1 year after vaccination with rabies virusa Treatments
Seroconversion prior to challenge
Seroconversion after challenge
Survivors/ total
DNA-i.m. DNA-gene gun Control
3/6 6/6 0/4
5/6 5/5b 4/4
5/6 3/5 1/4
a Monkeys were vaccinated i.m. with 100, 500, or 1000 g of DNA, or via gene gun with 8, 40, or 60 g of DNA; 375 days after vaccination the monkeys were challenged in the right and left masseter muscles with a salivary gland homogenate obtained from a rabid dog naturally-infected with a coyote rabies virus variant. b One rabies negative monkey died 3 days after viral challenge of age-related causes.
produced a minimal level of antibody after viral challenge. In contrast, the monkeys vaccinated via gene gun with either 60 or 40 g of DNA seroconverted after vaccination, and maintained high levels of antibody until euthanized (1:480 and 1:7680). Thus, 2/5 gene gun vaccinated, 1/6 i.m. vaccinated and 3/4 control animals died of rabies (Table 1).
4. Discussion Resistance to rabies has long been associated with the presence of neutralizing antibody [14]. Vaccine-induced antibody or passively administered immunoglobulin (for humans) is regarded as a key factor in prophylactic protection of animals and humans and post-exposure treatment of humans [14]. However, it has been shown on occasion that a correlation does not always exist between levels of neutralizing antibody in individual monkeys and their protection against death [15,16]. Antibody positive animals die and antibody negative animals survive. This suggests that a subtle balance may exist between survival and death
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during rabies virus infections [17]. One factor influencing this balance may be interferon. After exposure to rabies virus, interferon or interferon inducers, or a combination of a rabies vaccine with these reagents protect rabbits [18,19] and mice [20,21]. Furthermore, a reduction in mortality rate is detected in monkeys that had been given a rabies vaccine in combination with interferon or an interferon inducer, as compared to monkeys that received only vaccine [22]. It also has been determined that there is an early induction of natural killer cells following rabies DNA vaccination [23]. Virus specific cytotoxic T cells (CTL) might also be included as a factor associated with the balance of factors contributing to resistance to rabies virus. However, the importance of CTL in resistance to rabies virus remains unresolved. Some investigators believe that CTL are important for survival of mice against rabies [24,25], while others have shown with CD8+ and CD4+ T lymphocyte depletion studies that CTL are unimportant in resistance of mice to rabies [26]. The importance of CTL in the protection of non-human primates against rabies virus has not been studied. Non-human primates are known to be resistant to rabies virus [16,22,27]. Traditionally, the United States Department of Agriculture requires that all animal rabies vaccines currently licensed must protect at least 87% of the vaccinates against challenge in a controlled clinical trial, while at least 80% of non-vaccinates should develop rabies. Thus, an overwhelming challenge dose must be used to insure that 80% of the non-vaccinated controls die. As a consequence of the severe viral challenge, superior vaccination treatments are needed to protect monkeys. The efficacy assessment of human rabies vaccines also requires that the neutralizing antibody response is comparable to currently licensed vaccines. Although a protective antibody level has not been definitively determined, a titer of 0.5 IU/ml (1:20 titer in this study) is considered an adequate response and evidence of successful vaccination. In this study, neutralizing antibody was not detected after vaccination or viral challenge in the one i.m. vaccinated monkey that developed rabies. Two additional i.m. vaccinated monkeys also failed to seroconvert after immunization, but developed antibody after viral challenge and survived, suggesting that they had been immunologically primed by the vaccination. In contrast, all gene gun vaccinated animals developed antibody levels of at least 0.5 IU/ml after vaccination and produced excellent anamnestic antibody responses after viral challenge, but two died of rabies. Thus, we have the conundrum of two pre-challenge seronegative i.m. vaccinated animals that survived, two gene gun vaccinated monkeys that produced antibody after vaccination, but died, and an unvaccinated negative control that survived. There are numerous reports of unvaccinated rabies virus exposed animals resisting infection or recovering from infection [28–35]. Furthermore, it has been determined that the number of dogs recovering from experimental rabies infections may vary from 0 to 20%, and that recovery is independent of either virus strain or dosage [36]. The survival of the negative control monkey also raises questions concern-
ing possible susceptibility differences of out-bred monkeys against rabies virus. Notwithstanding the survival of the negative control, it is possible that the breakdown in protection of the vaccinated animals was aggravated by the three different times the monkeys were anesthetized with ketamine hydrochloride, handled and bled during the first 7 days of viral challenge. This procedure was the converse of our previous non-human primate experiment in which the DNA-vaccinated monkeys were not anesthetized, handled or bled after viral challenge, but 100% survived [8]. Interestingly, a single report suggests that stress and anesthetic drugs such as ketamine may interfere with immune responses during human post-exposure rabies treatment [37]. It also has been shown in rhesus monkeys and mice that general anaesthesia with ketamine is accompanied by a depressed antibody output [38], that T helper cell populations are depressed and suppressor cell populations are increased after surgical stress and the application of ketamine [39]. The handling-induced stress of animals also activates the hypothalmic-pituitary-adrenal axis leading to production of glucocorticoids that down regulate immune responses [40]. Furthermore, it has been suggested that stress associated with “handling” might be a mechanism for reactivation of latent rabies in carrier dogs [41]. Thus, the repeated ketamine treatments and the stress associated with handling and bleeding of the animals, might have tipped a subtle balance of resistance that is associated with neutralizing antibody. It also was determined herein that neutralizing antibody responses and protection against viral challenge did not correlate with the concentration of the single dose of DNA that was used for vaccination. For example, neither monkey vaccinated i.m. with 100 g of DNA made a primary antibody response, but both survived, whereas monkeys vaccinated i.m. with 1000 g of DNA made a primary antibody response and also survived. Furthermore, animals developing antibody after gene gun vaccination with 40 or 60 g of DNA either survived or died. In our previous study, all monkeys given 8 g primary and booster DNA vaccinations via gene gun produced neutralizing antibody and 100% survived. Although 8/11 (73%) DNA-vaccinated animals in this study survived viral challenge (5/6 i.m. vaccinated, and 3/5 gene gun vaccinated), the combined number of survivors of both groups, or the number of survivors of each group taken individually, was not statistically different from the 1/4 unvaccinated control animals that survived. Nonetheless, given this promising trend of resistance, repeating the experiments with less anesthesia, handling and bleeding of the animals after viral challenge, in addition to using larger sample sizes may result in statistical significance. Many different DNA vaccines elicit protection in small animal models. In larger species, in particular non-human primates, DNA vaccines are less effective. However, it has been shown that DNA vaccines protect macaques against rabies virus [8], HIV [42], dengue virus type 1 [43] and measles virus [44]. Interestingly, in all of these studies,
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booster DNA vaccinations were given to the monkeys prior to viral challenge. In contrast, in this study, the rhesus macaques were protected against rabies virus challenge more than 1 year after a DNA vaccination that did not include a subsequent booster DNA immunization. To our knowledge, this is the first demonstration of a one-time DNA vaccination protecting non-human primates against a human viral pathogen. In summary, we have shown that two different methods of rabies DNA vaccination of non-human primates elicited notably different neutralizing antibody responses. Irrespective of the neutralizing antibody response, or apparent lack of an antibody response, substantial protection against rabies virus challenge was demonstrated 1 year after i.m. or gene gun rabies DNA vaccination. This study demonstrates that a one-time rabies DNA vaccination has the potential for providing humans long-term protection against rabies virus.
Acknowledgements The authors 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, and the Animal Resources Branch, Centers for Disease Control, for care of the monkeys; G. Hettrick for graphic arts assistance; Nancy B. Ray for construction of the rabies DNA vaccine; and C. Rupprecht for providing the challenge rabies virus, and arranging for facilities to hold the rabies virus injected animals. We also thank the many professional staff at the CDC Viral and Rickettsial Zoonoses Branch, and the Animal Resources Branch, without whom this study could not have been possible. References [1] Fishbein DB, Robinson LE Rabies N Engl J Med 1993;329:1632–8. [2] Meslin F, Fishbien DB, Matter HC. Rationale and prospects for rabies elimination in developing countries. In: Rupprecht CE, Dietzschold B, Koprowski H, editors. Lyssaviruses. New York: Springer, 1994. p. 1–26. [3] Dressen DW. A global review of rabies vaccines for human use. Vaccine 1997;15(Suppl):S2–6. [4] Vodopija I, Clarke HF. Human vaccination against rabies. In: Baer GM, editor. The natural history of rabies. Boca Raton, FL: CRC Press, 1991. p. 571–95. [5] Gluck R, Kieny MP. Vaccination against rabies. In: Cryz SJ, editor. Vaccines and immunotherapy. Elmsford, NY: Pergamon Press, 1991. p. 246–55. [6] WHO Expert Committee on Rabies. Eighth report. World Health Organization Technical Report Series 1992;824:1–84. [7] Perrin P, Jacob Y, Tordo N. DNA-based immunization against lyssaviruses. Intervirology 2000;43:302–11. [8] Lodmell DL, Ray NB, Parnell MJ, Ewalt LC, Hanlon CA, Shaddock JH, et al. DNA immunization protects non-human primates against rabies virus. Nature Med 1998;4:949–52. [9] Ray NB, Ewalt LC, Lodmell DL. Nanogram quantities of plasmid DNA encoding the rabies virus glycoprotein protect mice against lethal rabies virus infection. Vaccine 1997;15:892–5.
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One-time gene gun or intramuscular rabies DNA vaccination of non-human primates: comparison of neutralizing antibody responses and protection against rabies virus 1 year after vaccination Donald L. Lodmell a,∗ , Michael J. Parnell a , John R. Bailey a , Larry C. Ewalt a , Cathleen A. Hanlon b a
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT 59840, USA b 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, GA 30333, USA Received 25 May 2001; received in revised form 28 August 2001; accepted 29 August 2001
Abstract We have previously shown that Macaca fascicularis (Cynomologus) monkeys receiving a primary and either one or two booster rabies DNA vaccinations are protected against rabies virus. In this study, we determined whether monkeys that had been vaccinated only once via gene gun or intramuscularly (i.m.) with different concentrations of DNA would be protected against rabies virus challenge. Neutralizing antibody responses were assayed for 1 year before the monkeys were challenged. Neutralizing antibody was detected at least 50 days earlier in gene gun vaccinated as compared to i.m. vaccinated animals. Prior to viral challenge, all (6/6, 100%) gene gun vaccinated animals, but only 3/6 (50%) i.m. vaccinated animals seroconverted. In general, antibody titers of the gene gun vaccinated animals were higher than the titers of the i.m. vaccinated animals. There was no correlation between the concentration of DNA used for vaccination, the neutralizing antibody responses elicited and protection against viral challenge. Seven days after viral challenge, a rapid and strong anamnestic antibody response was elicited in 100% of the gene gun vaccinated monkeys and in four i.m. vaccinated monkeys. Neutralizing antibody remained undetectable in two i.m. vaccinated monkeys. Overall, 60% (3/5) of the gene gun vaccinated animals and 87% (5/6) of the i.m. vaccinated monkeys survived viral challenge. This study is the first, to our knowledge, to show long-term protection of non-human primates against a human viral pathogen using a DNA vaccination protocol that did not include a booster immunization. Published by Elsevier Science Ltd. Keywords: Rabies virus; DNA vaccine; Protection; Neutralizing antibody
1. Introduction Rabies, ‘la rage’ or madness, has been the object of human fascination, torment and fear since the disease was recognized in antiquity [1]. The World Health Organization (WHO) estimates that 40,000–100,000 human deaths are caused by rabies each year [2], and that 10–12 million people in developing countries receive one or more doses of rabies vaccine annually after exposure to the virus [3]. Many developing countries continue to use vaccines produced in sheep, goat or suckling mouse brain, with subsequent inactivation of virus with ultraviolet light or phenol [4,5]. The WHO recommends that vaccines derived from brain tissue be discontinued and replaced with vaccines produced in tissue culture cells [6]. Although vaccines derived from cultured cells such as the human diploid cell vaccine (HDCV) ∗ Corresponding author. Tel.: +1-406-363-9360; fax: +1-406-363-9380. E-mail address: [email protected] (D.L. Lodmell).
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are efficacious and well tolerated, they are costly to produce and prohibitively expensive to purchase. DNA vaccines offer a new and powerful approach for the generation of rabies vaccines. The advantages of DNA vaccines are their ease of construction, high temperature stability, cost effectiveness and ability to induce a full-spectrum of humoral and cellular immune responses [7]. Recently, we showed that Macaca fascicularis (Cynomolgus) monkeys given a primary and either one or two booster vaccinations of a rabies DNA vaccine encoding the glycoprotein of the challenge virus standard strain are 100% protected against rabies virus challenge [8]. The monkeys had been vaccinated via gene gun, or with a combination of intradermal and intramuscular (i.m.) injections. In addition, antibody elicited by the DNA-vaccinated monkeys neutralized in vitro a worldwide spectrum of rabies virus variants, suggesting that a single DNA vaccine could be used globally against the classical rabies viruses [8].
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2. Materials and methods
with a total of 8, 40, or 60 g of DNA. DNA was coated onto 0.5 mg of gold beads 2.1 m in diameter, according to instructions provided by PowderJect Vaccines, Madison, WI, and administered with the Dermal PowderJect XR gene gun at 400 psi. The DNA was administered at 2 g per shot. A booster vaccination was not given. Blood was collected at regular intervals and sera were tested for rabies virus neutralizing antibody.
2.1. Plasmid construction
2.4. Neutralizing antibody assay
Construction of the pCMV4 plasmid DNA vaccine encoding the G of the challenge virus standard strain of rabies virus has been described [9,10].
Rabies virus neutralizing antibody titers were determined as previously described [11,12]. Reciprocal dilutions of individual sera that reduced the number of fluorescent foci 50% are shown in the figures. Antisera with known International Units (IU)/ml of rabies virus neutralizing antibody, a rabies hyperimmune mouse serum and the United States Standard Human Rabies Immunoglobulin R2, were included as positive controls in all 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.
In this study, we (1) compared for over 1 year, neutralizing antibody responses of monkeys that had been vaccinated one-time i.m., or via gene gun, with different concentrations of DNA and (2) determined whether the monkeys were resistant to rabies virus challenge 1 year after vaccination.
2.2. M. fascicularis (Cynomolgus) The M. fascicularis (Cynomolgus) colony at the Rocky Mountain Laboratories (RML) originated with captive monkeys from the Mauritius Islands. Fourteen animals were wild-caught stock (4–9 years in the RML colony), while two were captive-born at RML. The monkeys (12 female, 4 male), randomly assigned to experimental groups, ranged in age from 4–10 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 hydochloride (10 mg/kg i.m.) for inoculations and blood sampling. The animal facilities and animal care and use programs at the RML are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Both function in accordance with all United States Department of Agriculture, Department of Health and Human Services, and National Institutes of Health Regulations and Standards. The monkeys were housed indoors in artificial light (12/12 h dark/light cycle) and housed paired or singly in aluminum barred cages (15.1 ft2 floor space×63 in. high or 6.3 ft2 floor space × 32 in. high, respectively). The colony rooms are maintained at 21–26◦ C at 50% humidity. Prior to virus challenge, the monkeys were shipped to the Rabies Section, Centers for Disease Control (CDC), Atlanta, Georgia by a domestic commercial air freight provider, in full accordance with all United States Department of Agriculture, Department of Health and Human Services and National Institutes of Health Regulations and Standards. The husbandry of CDC animal facility is similar to that of the RML facility. 2.3. Vaccinated groups of monkeys Twelve monkeys (two animals per group) were vaccinated i.m. in the triceps muscle with either 100, 500, or 1000 g of DNA, or via gene gun above the axillary area of each upper arm and above the inguinal area of each upper thigh
2.5. Rabies virus challenge The monkeys were challenged 375 days after vaccination at the Rabies Section Unit, CDC, Atlanta, GA. For viral challenge, 0.5 ml of a 1:5 dilution of a salivary gland homogenate obtained from a rabid dog naturally infected with a coyote rabies virus variant [8,13] was injected in the left and right masseter muscles. The viral titer of the stock salivary gland homogenate was 106.5 mouse intracranial lethal dose50 /0.03 ml. Previously, challenge in the masseter muscles with the same concentration of the identical virus preparation caused rabies in 100% of the negative control monkeys [8]. After challenge, the monkeys were observed several times daily for clinical signs associated with rabies. At the first definitive clinical sign, animals were sedated and euthanized with a barbiturate solution administered intravenously. At necropsy, brain impressions were made and tested for rabies virus antigen by the direct fluorescent antibody test.
3. Results The i.m. and gene gun routes of vaccination elicited completely different neutralizing antibody responses (Fig. 1A and B). As early as 7 days after vaccination, 4/6 gene gun vaccinated animals were positive for antibody, and at 60 days 6/6 animals were positive. In contrast, neutralizing antibody was initially detected in two i.m. vaccinated animals 60 days after vaccination. A third i.m. vaccinated animal seroconverted at 180 days, but three i.m. vaccinated monkeys remained antibody negative. Furthermore, antibody levels of the two groups were notably different. The titers of the i.m. vaccinated animals never exceeded 1:80, whereas gene
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gun vaccinated animals attained levels as high as 1:1280. The elicitation of antibody in i.m. vaccinated animals also appeared to be DNA dose-dependent. Antibody was not detected in monkeys vaccinated with 100 g of DNA, whereas 3/4 animals vaccinated with 500 or 1000 g of DNA seroconverted. A dose-dependent response was not evident among the gene gun vaccinated animals. At the time of viral challenge, neutralizing antibody was present in 4/6 gene gun vaccinated and 3/6 i.m. vaccinated animals (Fig. 1A and B). Three days after viral challenge, one gene gun vaccinated rabies negative monkey died of age-related causes. Neutralizing antibody responses of the remaining animals were notably varied (Fig. 1A and B) (five animals were not
bled on day 3). Titers had increased in 2/2 gene gun and 2/2 i.m. vaccinated animals, but remained negative in 2/2 i.m. vaccinated monkeys. Seven days after challenge, all gene gun vaccinated animals had high (1:240) to very high (1:122,880) levels of neutralizing antibody. Among the i.m. vaccinated monkeys at this time, antibody was initially detected in one animal, and the levels of neutralizing antibody of the three previously positive animals had increased significantly (128–2048-fold). In addition, 2/4 negative controls developed antibody by day 7, and all (4/4) were seropositive by day 14, or the day of euthanasia. Three negative control animals developed rabies 12 and 13 days after viral challenge. The fourth negative control
Fig. 1. Neutralizing antibody responses and protection of monkeys challenged with rabies virus 1 year after DNA vaccination. (A) Intramuscular vaccination. Two monkeys were vaccinated i.m. in the triceps muscle with 100, 500 or 1000 g of DNA. (B) Gene gun vaccination. Two monkeys were vaccinated via gene gun with 8, 40, or 60 g of DNA above the axillary area of each arm and above the inguinal area of each thigh (2 g per shot). Three hundred seventy-five days after vaccination the monkeys were challenged i.m. in the masseter muscles with rabies virus. The animals were anesthetized with ketamine hyrochloride and bled at the designated intervals, and the sera tested for neutralizing antibody. The horizontal dashed line (1:20 titer) in both panels A and B indicates 0.5 IU/ml of neutralizing antibody, evidence of successful vaccination or immunologic priming. The arrow (↑) designates the day of rabies virus challenge. The numbers 1–4 above the non-vaccinated control animals (∗) identify each individual animal in this group. Data from the same control monkeys are shown in panels A and B. The day monkeys developed rabies and were euthanized is shown in parentheses. The antibody titer corresponds with the terminal bleed. Three days after viral challenge, five monkeys were not bled, and one gene gun vaccinated rabies negative monkey died of age-related causes.
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Fig. 1. (Continued).
remained clinically normal and seropositive up to 210 days after challenge, but was rabies negative at euthanasia. Three vaccinated animals also developed rabies and were euthanized on days 12 and 13. The animal vaccinated i.m. with 500 g of DNA did not seroconvert after vaccination, and Table 1 Summary of seroconversions and protection of non-human primates vaccinated one-time with a rabies DNA vaccine, and challenged 1 year after vaccination with rabies virusa Treatments
Seroconversion prior to challenge
Seroconversion after challenge
Survivors/ total
DNA-i.m. DNA-gene gun Control
3/6 6/6 0/4
5/6 5/5b 4/4
5/6 3/5 1/4
a Monkeys were vaccinated i.m. with 100, 500, or 1000 g of DNA, or via gene gun with 8, 40, or 60 g of DNA; 375 days after vaccination the monkeys were challenged in the right and left masseter muscles with a salivary gland homogenate obtained from a rabid dog naturally-infected with a coyote rabies virus variant. b One rabies negative monkey died 3 days after viral challenge of age-related causes.
produced a minimal level of antibody after viral challenge. In contrast, the monkeys vaccinated via gene gun with either 60 or 40 g of DNA seroconverted after vaccination, and maintained high levels of antibody until euthanized (1:480 and 1:7680). Thus, 2/5 gene gun vaccinated, 1/6 i.m. vaccinated and 3/4 control animals died of rabies (Table 1).
4. Discussion Resistance to rabies has long been associated with the presence of neutralizing antibody [14]. Vaccine-induced antibody or passively administered immunoglobulin (for humans) is regarded as a key factor in prophylactic protection of animals and humans and post-exposure treatment of humans [14]. However, it has been shown on occasion that a correlation does not always exist between levels of neutralizing antibody in individual monkeys and their protection against death [15,16]. Antibody positive animals die and antibody negative animals survive. This suggests that a subtle balance may exist between survival and death
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during rabies virus infections [17]. One factor influencing this balance may be interferon. After exposure to rabies virus, interferon or interferon inducers, or a combination of a rabies vaccine with these reagents protect rabbits [18,19] and mice [20,21]. Furthermore, a reduction in mortality rate is detected in monkeys that had been given a rabies vaccine in combination with interferon or an interferon inducer, as compared to monkeys that received only vaccine [22]. It also has been determined that there is an early induction of natural killer cells following rabies DNA vaccination [23]. Virus specific cytotoxic T cells (CTL) might also be included as a factor associated with the balance of factors contributing to resistance to rabies virus. However, the importance of CTL in resistance to rabies virus remains unresolved. Some investigators believe that CTL are important for survival of mice against rabies [24,25], while others have shown with CD8+ and CD4+ T lymphocyte depletion studies that CTL are unimportant in resistance of mice to rabies [26]. The importance of CTL in the protection of non-human primates against rabies virus has not been studied. Non-human primates are known to be resistant to rabies virus [16,22,27]. Traditionally, the United States Department of Agriculture requires that all animal rabies vaccines currently licensed must protect at least 87% of the vaccinates against challenge in a controlled clinical trial, while at least 80% of non-vaccinates should develop rabies. Thus, an overwhelming challenge dose must be used to insure that 80% of the non-vaccinated controls die. As a consequence of the severe viral challenge, superior vaccination treatments are needed to protect monkeys. The efficacy assessment of human rabies vaccines also requires that the neutralizing antibody response is comparable to currently licensed vaccines. Although a protective antibody level has not been definitively determined, a titer of 0.5 IU/ml (1:20 titer in this study) is considered an adequate response and evidence of successful vaccination. In this study, neutralizing antibody was not detected after vaccination or viral challenge in the one i.m. vaccinated monkey that developed rabies. Two additional i.m. vaccinated monkeys also failed to seroconvert after immunization, but developed antibody after viral challenge and survived, suggesting that they had been immunologically primed by the vaccination. In contrast, all gene gun vaccinated animals developed antibody levels of at least 0.5 IU/ml after vaccination and produced excellent anamnestic antibody responses after viral challenge, but two died of rabies. Thus, we have the conundrum of two pre-challenge seronegative i.m. vaccinated animals that survived, two gene gun vaccinated monkeys that produced antibody after vaccination, but died, and an unvaccinated negative control that survived. There are numerous reports of unvaccinated rabies virus exposed animals resisting infection or recovering from infection [28–35]. Furthermore, it has been determined that the number of dogs recovering from experimental rabies infections may vary from 0 to 20%, and that recovery is independent of either virus strain or dosage [36]. The survival of the negative control monkey also raises questions concern-
ing possible susceptibility differences of out-bred monkeys against rabies virus. Notwithstanding the survival of the negative control, it is possible that the breakdown in protection of the vaccinated animals was aggravated by the three different times the monkeys were anesthetized with ketamine hydrochloride, handled and bled during the first 7 days of viral challenge. This procedure was the converse of our previous non-human primate experiment in which the DNA-vaccinated monkeys were not anesthetized, handled or bled after viral challenge, but 100% survived [8]. Interestingly, a single report suggests that stress and anesthetic drugs such as ketamine may interfere with immune responses during human post-exposure rabies treatment [37]. It also has been shown in rhesus monkeys and mice that general anaesthesia with ketamine is accompanied by a depressed antibody output [38], that T helper cell populations are depressed and suppressor cell populations are increased after surgical stress and the application of ketamine [39]. The handling-induced stress of animals also activates the hypothalmic-pituitary-adrenal axis leading to production of glucocorticoids that down regulate immune responses [40]. Furthermore, it has been suggested that stress associated with “handling” might be a mechanism for reactivation of latent rabies in carrier dogs [41]. Thus, the repeated ketamine treatments and the stress associated with handling and bleeding of the animals, might have tipped a subtle balance of resistance that is associated with neutralizing antibody. It also was determined herein that neutralizing antibody responses and protection against viral challenge did not correlate with the concentration of the single dose of DNA that was used for vaccination. For example, neither monkey vaccinated i.m. with 100 g of DNA made a primary antibody response, but both survived, whereas monkeys vaccinated i.m. with 1000 g of DNA made a primary antibody response and also survived. Furthermore, animals developing antibody after gene gun vaccination with 40 or 60 g of DNA either survived or died. In our previous study, all monkeys given 8 g primary and booster DNA vaccinations via gene gun produced neutralizing antibody and 100% survived. Although 8/11 (73%) DNA-vaccinated animals in this study survived viral challenge (5/6 i.m. vaccinated, and 3/5 gene gun vaccinated), the combined number of survivors of both groups, or the number of survivors of each group taken individually, was not statistically different from the 1/4 unvaccinated control animals that survived. Nonetheless, given this promising trend of resistance, repeating the experiments with less anesthesia, handling and bleeding of the animals after viral challenge, in addition to using larger sample sizes may result in statistical significance. Many different DNA vaccines elicit protection in small animal models. In larger species, in particular non-human primates, DNA vaccines are less effective. However, it has been shown that DNA vaccines protect macaques against rabies virus [8], HIV [42], dengue virus type 1 [43] and measles virus [44]. Interestingly, in all of these studies,
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booster DNA vaccinations were given to the monkeys prior to viral challenge. In contrast, in this study, the rhesus macaques were protected against rabies virus challenge more than 1 year after a DNA vaccination that did not include a subsequent booster DNA immunization. To our knowledge, this is the first demonstration of a one-time DNA vaccination protecting non-human primates against a human viral pathogen. In summary, we have shown that two different methods of rabies DNA vaccination of non-human primates elicited notably different neutralizing antibody responses. Irrespective of the neutralizing antibody response, or apparent lack of an antibody response, substantial protection against rabies virus challenge was demonstrated 1 year after i.m. or gene gun rabies DNA vaccination. This study demonstrates that a one-time rabies DNA vaccination has the potential for providing humans long-term protection against rabies virus.
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