Saturday
13
June
1992
No 8807
ORIGINAL ARTICLES Immunisation with canarypox virus rabies glycoprotein
expressing
Poxviruses have many useful features as vectors for genes that carry immunising antigens from other viruses, such as ease of production and induction of cellular and humoral immunity, but there is concern about the safety of vaccinia virus. We turned to an avian poxvirus (canarypox); this virus undergoes abortive replication in mammalian cells that enables presentation of early gene products to the immune system. Canarypox virus was used as a vector for the rabies glycoprotein G gene. The safety and efficacy of the recombinant (ALVAC-RG; vCP65) were tested in several animal species, then it was subjected to a phase 1 clinical trial. Twenty-five volunteers were randomly assigned to subcutaneous injections of the recombinant (three groups [A, B, and C] received two doses each of 103·5, 104·5, and 105·5 tissue-culture infectious doses50, respectively) or of human diploid cell culture vaccine (HDC; 6·52 international potency units per dose). 28 days after the second dose, all nine ALVAC-RG group-C subjects and two of three group-B subjects had rabies neutralising antibody concentrations of at least 0·5 IU/ml, the level associated with protection in animals. Although the geometric mean titre of these antibodies at that time was lower in group C than in the ten H DC recipients (4·4 [range 0·9-12·5] vs 11·5 [4·7-25·3] IU/ml), a single booster dose at 6 months induced a recall response in volunteers primed with either vaccine. Side-effects associated with ALVAC-RG were mild and of short duration and occurred at similar frequency to those of HDC vaccine. This study has shown the potential of nonreplicating poxviruses as vectors for vaccination in human beings. Trials of canarypox-virus recombinants at higher doses and by other routes of administration are needed.
produced with this vector include a vaccinia rabies recombinant, used for oral vaccination of wild animals against rabies.4 Vaccinia virus has been given to millions of people, but developers of new vaccines are reluctant to use it because it causes complications rarely in healthy subjects and more frequently among immunosuppressed people. However, the poxviruses have many advantages, including ease of production in high titre, induction of cellular immune responses, and priming of immunity.’ We turned to avian poxviruses, which share the advantages of other poxvirus vectors without the associated safety difficulties; although incapable of complete replication in mammalian cells, avian poxviruses undergo abortive replication during which early genes, including properly inserted ones, are transcribed and translated, resulting in presentation of protein to the immune system.6-11 We chose a poxvirus of
Introduction
ADDRESSES: Pasteur-Mérieux Serums et Vaccins, Lyon (M. Cadoz, MD, B. Meignier, DVM, S. Plotkin, MD); Centre Hospitalier Régional et Universitaire de Reims, France (A. Strady, MD); and Virogenetics, Troy, New York, USA (J. Taylor, PhD, J. Tartaglia, PhD, E. Paoletti, PhD). Correspondence to Dr Michel Cadoz, Pasteur-Mérieux Serums et Vaccins, 3 avenue Pasteur, 92430 Marnes-la-Coquette, France.
One of the
important recent steps in vaccine the introduction of vectors to carry genes development that code for immunising antigens.1 Vaccinia virus was among the first vectors to be used2°3 and the many vaccines most
was
canaries and evaluated its usefulness as a vector with the gene for rabies glycoprotein Gl2-14 in animals and in a phase 1 clinical trial.
Subjects and methods The methods used to produce ALVAC-RG (vCP65) have been described previously.2,6 The vector (ALVAC) was plaque purified from the live attenuated vaccine strain against canarypox (Kanapox, Rhone-Merieux, Lyon, France). The cDNA coding for the ERA strain rabies glycoprotein G" was inserted in place of the C5 locus under the control of the vaccinia virus H6 promoter.15-17 The recombinant was subjected to six rounds of plaque purification. No change was detected in the DNA of the G gene or in the ALVAC flanking sequences. The expression of the insert was shown by immunoprecipitation of lysates of ALVAC-RG-infected cells (chick embryo fibroblasts, Vero, MRC5) with a monoclonal antibody specific for rabies G glycoprotein and by standard immunofluorescence techniques. Vaccine was produced in primary chick embryo fibroblasts, derived from pathogen-free eggs, infected with 0 01 tissue-culture infectious doses (TCID per cell. The vaccine was made from a clarified lysate of infected cells in serum-free medium; lyophilisation stabiliser was added before freeze-drying. Batches of decreasing titres were prepared by ten-fold dilution. The vaccines and other ingredients showed no undesirable features in standard
quality-control assays.
1430
TABLE I-CLINICAL REACTIONS IN 5 DAYS AFTER INJECTIONS 1 AND 2
In vitro Vero and MRC5 cells did not support growth of ALVAC-RG, even after eight or ten blind serial passages. Human cell lines (MRC-5, WISH, Detroit 532, HEL, HNK, or EpsteinBarr-virus-transformed lymphoblastoid) infected with ALVAC-
RG showed no accumulation of virus-specific DNA. The safety and efficacy of ALVAC-RG were tested in canaries, chickens, suckling and adult mice, hamsters, guineapigs, rabbits, cats, dogs, squirrel monkeys, rhesus macaques, and chimpanzees inoculated with doses ranging from 105 to 108 TCID50’ Several
ALVAC-RG; subgroup 3 two doses of ALVAC-RG then HDC; and subgroup 4 three doses of ALVAC-RG. Subjects were monitored for 1 h after injection then examined daily for the next 5 days. They were asked to record local and systemic reactions for the next 3 weeks and were questioned by telephone twice a week. Blood samples were taken before enrolment and 2, 4, and 6 days after each injection. Analysis included complete blood cell count and liver enzyme and creatine kinase assays.
Antibody assays were done 7 days before the first injection and 7, 28, 35, 56, 173, 187, and 208 days afterwards. Neutralising
commonly subcutaneous, intramuscular, and intradermal but also oral (monkeys and mice) and intracerebral (mice). In canaries, ALVAC-RG caused a lesion at the site of scarification but there were no signs of disease and no deaths. No side-effect was seen in any of the animal safety tests except regressive induration and superficial necrosis in rabbits injected intradermally. Inoculation of ALVAC-RG in rodents, dogs, cats, and primates induced rabies neutralising antibody detectable by the rapid fluorescent focus inhibition test (RFFIT).18 Protection was shown in mice, cats, and dogs. We enrolled for the phase 1 trial twenty-five volunteers, aged 20-50 years, found to be healthy by complete medical history, physical examination, and a haematological and blood chemistry analysis. Reasons for exclusion were pregnancy, allergies, immune depression of any kind, chronic debilitating disease, cancer, rabies immunisation, injection of immunoglobulins in the previous 3 months, and seropositivity to human immunodeficiency virus or to hepatitis B virus surface antigen. Participants were randomly assigned either standard human diploid cell culture rabies vaccine (HDC; batch no E0751, 6-52 international potency units; PasteurMerieux) or the study vaccine. Three batches of ALVAC-RG with titres of 1035, 10", and 10"TCID50 per dose, were used sequentially in three groups of volunteers (groups A, B, and C, respectively). Each volunteer received two injections of the same vaccine subcutaneously in the deltoid region with an interval of 4 weeks. The investigators but not the participants were aware which type of vaccine was being used. Subjects in group C (highest dose of ALVAC-RG) and the HDC group were given booster doses 6 months after the first course of immunisation; they were randomly assigned either the same vaccine as previously or the other vaccine. As a result four subgroups were formed: subgroup 1 received three doses of HDC in all; subgroup 2 two doses of HDC then
The mean age of the ten male and fifteen female volunteers was 32 (218) years. All but three subjects had evidence of previous smallpox vaccination (typical scar, vaccination history, or both). There were three subjects in each of the lower-dose groups (A and B), nine in group C (including the three who had not received smallpox vaccination), and ten in the HDC group. During the first immunisation series, the only systemic reactions attributable to vaccination were fever above 377°C within 24 h of injection in two subjects (one HDC, one group C; table I). There were local reactions in nine recipients of HDC vaccine, in one group-B subject, and in all nine group-C subjects; no group-A subject showed a local reaction. Tenderness was the most common symptom and was always mild. Other local symptoms included redness and induration, which were also mild and transient. Most symptoms subsided within 24 h and none lasted more
TABLE t!—CONCENTRATIONS OF RABIES NEUTRALISING ANTIBODIES
(NA)
routes were
used,
most
rabies virus were sought with the RFFIT.15 antibodies were measured by direct enzymelinked immunosorbent assay. The antigen, a suspension of purified canarypox-virus disrupted with 0-% Triton X100, was coated on microplates. Fixed dilutions of the serum samples were incubated in the plates for 2 h at room temperature and reacting antibodies were revealed with a peroxidase-labelled goat antibody to human IgG. The results are expressed as the optical density at 490 nm. antibodies
Results
VACCINATION
Times
are
from first vaccination.
*Optical density at 1/25 dilution
GMT=geometnc
mean
titre; ND= not done
to
Canarypox-virus
AND CANARYPOX-VIRUS ANTIBODIES BEFORE AND AFTER
1431
than 72 h. There was no significant change in blood cell counts or liver enzyme or creatine kinase activities. 28 days after the first injection all HDC recipients had concentrations of rabies neutralising antibodies of 05 IU/ml or higher, whereas no subject in group A or B and only two subjects in group C had this concentration (table 11). At day 56 (ie, 28 days after the second injection) two group-B subjects and all nine group-C subjects had neutralising antibodies at 0-5 IU/ml or greater; still no group-A subject had achieved this level. The geometric mean concentration was significantly higher in the HDC group than in group C (p=0007, Student’s t test). At day 180, rabies antibody concentrations had fallen substantially in all subjects but were still above 0-5 IU/ml in five HDC recipients and in five group-C subjects; the geometric mean values did not differ significantly (0-51 vs 0-45 IU/ml). Baseline values of canarypox-virus antibodies varied from 0.22 to 1-23 OD units (table 11) even though subjects with the highest titres could not recall contact with canary birds. Serocoversion (defined as a more than two-fold increase SUBGROUP1
HDC
1
SUBGROUP22
ALVAC-RG
i
4 SUBGROUP4
ALVAC-RG
i
Time from first
injection (days)
Rabies neutralising antibody titres after booster injections of HDC
or
ALVAC-RG.
from baseline to after the second injection) occurred in no group-A or HDC subject, in one group-B subject, and in all nine group-C subjects; group C included the three patients with no evidence of previous smallpox vaccination. The vaccines were again well tolerated at the time of the booster injection; two of nine HDC booster recipients and one of ten ALVAC-RG booster recipients had fever and five and six, respectively, had local reactions. The booster dose led to a further increase in rabies antibodies in every subject, in all vaccine subgroups (figure). Overall, however, the ALVAC-RG booster elicited lower responses than the HDC booster and subgroup 4 (who received three doses of ALVAC-RG) had lower antibody concentrations than the other three groups. The ALVAC-RG booster injection resulted in an increase in canarypox-virus antibodies in three of five subjects who had previously received HDC vaccine and in all five subjects who had previously received ALVAC-RG.
Discussion The ALVAC-RG vaccine used in this trial was safe and well tolerated. The most common side-effects, mild tenderness sometimes associated with local redness and induration, occurred at similar frequency with the licensed HDC rabies vaccine and did not suggest local replication of the canarypox-virus. Nevertheless, substantial amounts of antibodies to both the canarypox-virus vector and the expressed rabies glycoprotein were induced. In mice, the presence of rabies neutralising antibodies at more than 0-5 IU/ml correlates well with the seroneutralisation test. This level of antibody response was achieved with two injections in all recipients of the highest dose of ALVAC-RG and in two of three recipients of the intermediate dose. Both vaccines were given by the subcutaneous route as recommended for live vaccines; this factor could explain the late appearance of antibodies in HDC recipients.1921 The route of choice for the recombinant vaccine should be further assessed. The geometric mean rabies neutralising antibody concentration was lower after ALVAC-RG than after HDC but still well above the minimum associated with protection in animals. The clear dose-response effect suggests that an even higher dose than we used might induce a stronger response. Cellular immune responses, not measured in this initial trial, will be studied in future. Another important finding was that pre-existing immunity elicited by either the canarypox-virus vector or the rabies glycoprotein had no blocking effect on responses to booster vaccination with either vaccine. This finding contrasts with those on vaccinia-virus recombinants, in which the immune response seems to be blocked by pre-existing immunisation against smallpox.521 We have shown that a non-replicating poxvirus can serve as an immunising vector in human beings, with all the advantages that replicating agents confer on the immune response, but with greater safety than a fully permissive virus. Future developments will include improvement of canarypox-virus growth in tissue culture, larger-scale tests of safety and immunogenicity, evaluation of immune responses to higher doses, and clinical trials of other canarypox-virus recombinants that have already given promising results in animals. We thank Mr Bernard Fanget for the preparation of the lots of study vaccine, Mrs Catherine Gerdil and Mrs Therese-Marie Jourdier for
serological assays, and Dr Christine clinical assistance.
Rouger
and Mrs Annette Haas for
1432
REFERENCES 1. WHO meeting, Geneva, June 19-22, 1989. Potential use of live viral and bacterial vectors for vaccines. Vaccine 1990; 8: 425-37. 2. Panicali D, Paoletti E. Construction of poxviruses as cloning vectors/ insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc Natl Acad Sci USA 1982; 79: 4927-31. 3. Mackett M, Smith GL, Moss B. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci USA 1982; 79: 7415-19. 4. Brochier B, Kieny MP, Costy F, et al. Large-scale eradication of rabies using recombinant vaccinia-rabies vaccine. Nature 1991; 354: 520-22. 5. Cooney EL, Collier AC, Greenberg PD, et al. Safety and immunological response to a recombinant vaccinia virus vaccine expressing HIV envelope glycoprotein. Lancet 1991; 337: 567-72. 6. Taylor J, Weinberg R, Lanquet B, et al. Recombinant fowlpox virus inducing protective immunity in non-avian species. Vaccine 1988; 6: 497-503. 7. Taylor J, Weinberg R, Kawaoka Y, et al. Protective immunity against avian influenza induced by a fowlpox virus recombinant. Vaccine 1988; 6: 504-08. 8. Taylor J, Paoletti E. Fowlpox virus as a vector in non-avian species. Vaccine 1988; 6: 466-68. 9. Taylor J, Weinberg R, et al. Non-replicating viral vectors as potential vaccines: recombinant canarypox virus expressing measles virus fusion (F) and hemagglutinin (HA) glycoproteins. Virology 1992; 187: 321-28. 10. Wild F, Giraudon P, Spehner D, et al. Fowlpox virus recombinant encoding the measles virus fusion protein: protection of mice against fatal measles encephalitis. Vaccine 1990; 8: 441-42.
11. Baxby D, Paoletti E. Potential use of non-replicating vectors as recombinant vaccines. Vaccine 1992; 10: 8-9. 12. Taylor J, Trimarchi C, Weinberg R, et al. Efficacy studies on a canarypox-rabies recombinant virus. Vaccine 1991; 9: 190-93. 13. Wiktor TJ, Macfarlan RI, Reagan KJ, et al. Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene. Proc Natl Acad Sci USA
14.
1984; 81: 7194-98.
Kieny MP, Lathe R, Drillien R, glycoprotein from a recombinant
al. Expression of rabies virus vaccinia virus. Nature 1984; 312:
et
163-66. 15. Smith JS,
Yager PA. A rapid tissue culture test for determining rabies neutralizing antibody. In: Kaplan MM, Koprowski H. Laboratory techniques in rabies. Geneva: WHO Monogr Series, 1973: 23. 16. Piccini A, Perkus ME, Paoletti E. Vaccinia virus as an expression vector. Methods Enzymol 1987; 153: 545-63. 17. Goebel SJ, Johnson GP, Perkus ME, et al. The complete DNA sequence of vaccinia virus. Virology 1990; 179: 247-66. 18. Goebel SJ, Johnson GP, Perkus ME, et al. Appendix to "The complete DNA sequence of vaccinia virus". Virology 1990; 179: 517-63. 19. Klietmann W, Schottle A, Kleitmann B, et al. A large scale antirabies immunization study in humans using HDCS vaccine: prophylactic vaccination using different routes of application and post-exposure treatment combined with and without simultaneous serum administration. In: Kuwert E, Wiktor T, Koprowski H, eds. Cell culture rabies vaccines and their protection effect in man. Geneva: International Green Cross, 1981: 330-37. 20. Kuwert EK, Barsenbach C, Wemer J, et al. Early/high and late/low responders among HDCS vaccinees. In: Kuwert EK, Wiktor T, Koprowski H, eds. Cell culture rabies vaccines and their protection effect in man. Geneva: International Green Cross, 1981: 160-67. 21. Etlinger HM, Altenberger W. Overcoming inhibition of antibody responses to a malaria recombinant vaccinia virus caused by prior exposure to wild type virus. Vaccine 1991; 9: 470-72.
Double-blind crossover comparison of human and porcine insulins in patients reporting lack of
hypoglycaemia awareness
There has been much debate about reports that insulin-treated diabetic patients lose of hypoglycaemic symptoms on awareness changing from porcine to human insulin. In a
some
double-blind,
crossover
study,
we
sought
differences between porcine and human insulin in the frequency and characteristics of hypoglycaemic episodes among patients who reported a reduction of awareness of hypoglycaemia after changing treatment.
We studied 50 patients referred by their physicians because of complaints of lack of awareness of hypoglycaemia on human insulin. They had had diabetes for a mean of 20 (SD 12) years and 70% had good or acceptable glycaemic control. Each patient was treated in a double-blind manner for four 1-month periods, two with human and two with porcine insulin, in random order. Only 2 patients correctly identified the sequence of insulin treatments used; 8 or 9 would have been expected to do so by chance alone. The mean percentage of hypoglycaemic episodes associated with reduced or absent awareness was 64% (SD 30%) for human insulin and 69% (31%) for porcine insulin.
We could find no statistically significant differences between the insulin species with respect to glycaemic control or the frequency, timing, severity, or awareness of hypoglycaemia. Reduced hypoglycaemia awareness is common with both human and porcine insulins.
Introduction In 1987 Berger and colleagues! reported a reduction in hunger and sweating in a group of patients with insulindependent diabetes after a change in treatment from porcine to human insulin. This report led to speculation that hypoglycaemia induced by human insulin was associated with less sympatheticoadrenal activation than that induced by animal insulins. Subsequent conflicting findings on the
ADDRESSES: Department of Endocrinology, Diabetes, and Metabolism, Prince of Wales Hospital, Randwick (S. Colagiuri, FRACP); Novo Nordisk Pharmaceuticals, North Rocks (J J. Miller, PhD); and School of Mathematical Sciences, University of Technology, Sydney (P. Petocz, PhD), New South Wales, Australia. Correspondence to Dr Stephen Colagiuri, Department of Endocrinology, Diabetes, and Metabolism, Prince of Wales Hospital, High Street, Randwick, New South Wales 2031, Australia.
13
June
1992
No 8807
ORIGINAL ARTICLES Immunisation with canarypox virus rabies glycoprotein
expressing
Poxviruses have many useful features as vectors for genes that carry immunising antigens from other viruses, such as ease of production and induction of cellular and humoral immunity, but there is concern about the safety of vaccinia virus. We turned to an avian poxvirus (canarypox); this virus undergoes abortive replication in mammalian cells that enables presentation of early gene products to the immune system. Canarypox virus was used as a vector for the rabies glycoprotein G gene. The safety and efficacy of the recombinant (ALVAC-RG; vCP65) were tested in several animal species, then it was subjected to a phase 1 clinical trial. Twenty-five volunteers were randomly assigned to subcutaneous injections of the recombinant (three groups [A, B, and C] received two doses each of 103·5, 104·5, and 105·5 tissue-culture infectious doses50, respectively) or of human diploid cell culture vaccine (HDC; 6·52 international potency units per dose). 28 days after the second dose, all nine ALVAC-RG group-C subjects and two of three group-B subjects had rabies neutralising antibody concentrations of at least 0·5 IU/ml, the level associated with protection in animals. Although the geometric mean titre of these antibodies at that time was lower in group C than in the ten H DC recipients (4·4 [range 0·9-12·5] vs 11·5 [4·7-25·3] IU/ml), a single booster dose at 6 months induced a recall response in volunteers primed with either vaccine. Side-effects associated with ALVAC-RG were mild and of short duration and occurred at similar frequency to those of HDC vaccine. This study has shown the potential of nonreplicating poxviruses as vectors for vaccination in human beings. Trials of canarypox-virus recombinants at higher doses and by other routes of administration are needed.
produced with this vector include a vaccinia rabies recombinant, used for oral vaccination of wild animals against rabies.4 Vaccinia virus has been given to millions of people, but developers of new vaccines are reluctant to use it because it causes complications rarely in healthy subjects and more frequently among immunosuppressed people. However, the poxviruses have many advantages, including ease of production in high titre, induction of cellular immune responses, and priming of immunity.’ We turned to avian poxviruses, which share the advantages of other poxvirus vectors without the associated safety difficulties; although incapable of complete replication in mammalian cells, avian poxviruses undergo abortive replication during which early genes, including properly inserted ones, are transcribed and translated, resulting in presentation of protein to the immune system.6-11 We chose a poxvirus of
Introduction
ADDRESSES: Pasteur-Mérieux Serums et Vaccins, Lyon (M. Cadoz, MD, B. Meignier, DVM, S. Plotkin, MD); Centre Hospitalier Régional et Universitaire de Reims, France (A. Strady, MD); and Virogenetics, Troy, New York, USA (J. Taylor, PhD, J. Tartaglia, PhD, E. Paoletti, PhD). Correspondence to Dr Michel Cadoz, Pasteur-Mérieux Serums et Vaccins, 3 avenue Pasteur, 92430 Marnes-la-Coquette, France.
One of the
important recent steps in vaccine the introduction of vectors to carry genes development that code for immunising antigens.1 Vaccinia virus was among the first vectors to be used2°3 and the many vaccines most
was
canaries and evaluated its usefulness as a vector with the gene for rabies glycoprotein Gl2-14 in animals and in a phase 1 clinical trial.
Subjects and methods The methods used to produce ALVAC-RG (vCP65) have been described previously.2,6 The vector (ALVAC) was plaque purified from the live attenuated vaccine strain against canarypox (Kanapox, Rhone-Merieux, Lyon, France). The cDNA coding for the ERA strain rabies glycoprotein G" was inserted in place of the C5 locus under the control of the vaccinia virus H6 promoter.15-17 The recombinant was subjected to six rounds of plaque purification. No change was detected in the DNA of the G gene or in the ALVAC flanking sequences. The expression of the insert was shown by immunoprecipitation of lysates of ALVAC-RG-infected cells (chick embryo fibroblasts, Vero, MRC5) with a monoclonal antibody specific for rabies G glycoprotein and by standard immunofluorescence techniques. Vaccine was produced in primary chick embryo fibroblasts, derived from pathogen-free eggs, infected with 0 01 tissue-culture infectious doses (TCID per cell. The vaccine was made from a clarified lysate of infected cells in serum-free medium; lyophilisation stabiliser was added before freeze-drying. Batches of decreasing titres were prepared by ten-fold dilution. The vaccines and other ingredients showed no undesirable features in standard
quality-control assays.
1430
TABLE I-CLINICAL REACTIONS IN 5 DAYS AFTER INJECTIONS 1 AND 2
In vitro Vero and MRC5 cells did not support growth of ALVAC-RG, even after eight or ten blind serial passages. Human cell lines (MRC-5, WISH, Detroit 532, HEL, HNK, or EpsteinBarr-virus-transformed lymphoblastoid) infected with ALVAC-
RG showed no accumulation of virus-specific DNA. The safety and efficacy of ALVAC-RG were tested in canaries, chickens, suckling and adult mice, hamsters, guineapigs, rabbits, cats, dogs, squirrel monkeys, rhesus macaques, and chimpanzees inoculated with doses ranging from 105 to 108 TCID50’ Several
ALVAC-RG; subgroup 3 two doses of ALVAC-RG then HDC; and subgroup 4 three doses of ALVAC-RG. Subjects were monitored for 1 h after injection then examined daily for the next 5 days. They were asked to record local and systemic reactions for the next 3 weeks and were questioned by telephone twice a week. Blood samples were taken before enrolment and 2, 4, and 6 days after each injection. Analysis included complete blood cell count and liver enzyme and creatine kinase assays.
Antibody assays were done 7 days before the first injection and 7, 28, 35, 56, 173, 187, and 208 days afterwards. Neutralising
commonly subcutaneous, intramuscular, and intradermal but also oral (monkeys and mice) and intracerebral (mice). In canaries, ALVAC-RG caused a lesion at the site of scarification but there were no signs of disease and no deaths. No side-effect was seen in any of the animal safety tests except regressive induration and superficial necrosis in rabbits injected intradermally. Inoculation of ALVAC-RG in rodents, dogs, cats, and primates induced rabies neutralising antibody detectable by the rapid fluorescent focus inhibition test (RFFIT).18 Protection was shown in mice, cats, and dogs. We enrolled for the phase 1 trial twenty-five volunteers, aged 20-50 years, found to be healthy by complete medical history, physical examination, and a haematological and blood chemistry analysis. Reasons for exclusion were pregnancy, allergies, immune depression of any kind, chronic debilitating disease, cancer, rabies immunisation, injection of immunoglobulins in the previous 3 months, and seropositivity to human immunodeficiency virus or to hepatitis B virus surface antigen. Participants were randomly assigned either standard human diploid cell culture rabies vaccine (HDC; batch no E0751, 6-52 international potency units; PasteurMerieux) or the study vaccine. Three batches of ALVAC-RG with titres of 1035, 10", and 10"TCID50 per dose, were used sequentially in three groups of volunteers (groups A, B, and C, respectively). Each volunteer received two injections of the same vaccine subcutaneously in the deltoid region with an interval of 4 weeks. The investigators but not the participants were aware which type of vaccine was being used. Subjects in group C (highest dose of ALVAC-RG) and the HDC group were given booster doses 6 months after the first course of immunisation; they were randomly assigned either the same vaccine as previously or the other vaccine. As a result four subgroups were formed: subgroup 1 received three doses of HDC in all; subgroup 2 two doses of HDC then
The mean age of the ten male and fifteen female volunteers was 32 (218) years. All but three subjects had evidence of previous smallpox vaccination (typical scar, vaccination history, or both). There were three subjects in each of the lower-dose groups (A and B), nine in group C (including the three who had not received smallpox vaccination), and ten in the HDC group. During the first immunisation series, the only systemic reactions attributable to vaccination were fever above 377°C within 24 h of injection in two subjects (one HDC, one group C; table I). There were local reactions in nine recipients of HDC vaccine, in one group-B subject, and in all nine group-C subjects; no group-A subject showed a local reaction. Tenderness was the most common symptom and was always mild. Other local symptoms included redness and induration, which were also mild and transient. Most symptoms subsided within 24 h and none lasted more
TABLE t!—CONCENTRATIONS OF RABIES NEUTRALISING ANTIBODIES
(NA)
routes were
used,
most
rabies virus were sought with the RFFIT.15 antibodies were measured by direct enzymelinked immunosorbent assay. The antigen, a suspension of purified canarypox-virus disrupted with 0-% Triton X100, was coated on microplates. Fixed dilutions of the serum samples were incubated in the plates for 2 h at room temperature and reacting antibodies were revealed with a peroxidase-labelled goat antibody to human IgG. The results are expressed as the optical density at 490 nm. antibodies
Results
VACCINATION
Times
are
from first vaccination.
*Optical density at 1/25 dilution
GMT=geometnc
mean
titre; ND= not done
to
Canarypox-virus
AND CANARYPOX-VIRUS ANTIBODIES BEFORE AND AFTER
1431
than 72 h. There was no significant change in blood cell counts or liver enzyme or creatine kinase activities. 28 days after the first injection all HDC recipients had concentrations of rabies neutralising antibodies of 05 IU/ml or higher, whereas no subject in group A or B and only two subjects in group C had this concentration (table 11). At day 56 (ie, 28 days after the second injection) two group-B subjects and all nine group-C subjects had neutralising antibodies at 0-5 IU/ml or greater; still no group-A subject had achieved this level. The geometric mean concentration was significantly higher in the HDC group than in group C (p=0007, Student’s t test). At day 180, rabies antibody concentrations had fallen substantially in all subjects but were still above 0-5 IU/ml in five HDC recipients and in five group-C subjects; the geometric mean values did not differ significantly (0-51 vs 0-45 IU/ml). Baseline values of canarypox-virus antibodies varied from 0.22 to 1-23 OD units (table 11) even though subjects with the highest titres could not recall contact with canary birds. Serocoversion (defined as a more than two-fold increase SUBGROUP1
HDC
1
SUBGROUP22
ALVAC-RG
i
4 SUBGROUP4
ALVAC-RG
i
Time from first
injection (days)
Rabies neutralising antibody titres after booster injections of HDC
or
ALVAC-RG.
from baseline to after the second injection) occurred in no group-A or HDC subject, in one group-B subject, and in all nine group-C subjects; group C included the three patients with no evidence of previous smallpox vaccination. The vaccines were again well tolerated at the time of the booster injection; two of nine HDC booster recipients and one of ten ALVAC-RG booster recipients had fever and five and six, respectively, had local reactions. The booster dose led to a further increase in rabies antibodies in every subject, in all vaccine subgroups (figure). Overall, however, the ALVAC-RG booster elicited lower responses than the HDC booster and subgroup 4 (who received three doses of ALVAC-RG) had lower antibody concentrations than the other three groups. The ALVAC-RG booster injection resulted in an increase in canarypox-virus antibodies in three of five subjects who had previously received HDC vaccine and in all five subjects who had previously received ALVAC-RG.
Discussion The ALVAC-RG vaccine used in this trial was safe and well tolerated. The most common side-effects, mild tenderness sometimes associated with local redness and induration, occurred at similar frequency with the licensed HDC rabies vaccine and did not suggest local replication of the canarypox-virus. Nevertheless, substantial amounts of antibodies to both the canarypox-virus vector and the expressed rabies glycoprotein were induced. In mice, the presence of rabies neutralising antibodies at more than 0-5 IU/ml correlates well with the seroneutralisation test. This level of antibody response was achieved with two injections in all recipients of the highest dose of ALVAC-RG and in two of three recipients of the intermediate dose. Both vaccines were given by the subcutaneous route as recommended for live vaccines; this factor could explain the late appearance of antibodies in HDC recipients.1921 The route of choice for the recombinant vaccine should be further assessed. The geometric mean rabies neutralising antibody concentration was lower after ALVAC-RG than after HDC but still well above the minimum associated with protection in animals. The clear dose-response effect suggests that an even higher dose than we used might induce a stronger response. Cellular immune responses, not measured in this initial trial, will be studied in future. Another important finding was that pre-existing immunity elicited by either the canarypox-virus vector or the rabies glycoprotein had no blocking effect on responses to booster vaccination with either vaccine. This finding contrasts with those on vaccinia-virus recombinants, in which the immune response seems to be blocked by pre-existing immunisation against smallpox.521 We have shown that a non-replicating poxvirus can serve as an immunising vector in human beings, with all the advantages that replicating agents confer on the immune response, but with greater safety than a fully permissive virus. Future developments will include improvement of canarypox-virus growth in tissue culture, larger-scale tests of safety and immunogenicity, evaluation of immune responses to higher doses, and clinical trials of other canarypox-virus recombinants that have already given promising results in animals. We thank Mr Bernard Fanget for the preparation of the lots of study vaccine, Mrs Catherine Gerdil and Mrs Therese-Marie Jourdier for
serological assays, and Dr Christine clinical assistance.
Rouger
and Mrs Annette Haas for
1432
REFERENCES 1. WHO meeting, Geneva, June 19-22, 1989. Potential use of live viral and bacterial vectors for vaccines. Vaccine 1990; 8: 425-37. 2. Panicali D, Paoletti E. Construction of poxviruses as cloning vectors/ insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc Natl Acad Sci USA 1982; 79: 4927-31. 3. Mackett M, Smith GL, Moss B. Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci USA 1982; 79: 7415-19. 4. Brochier B, Kieny MP, Costy F, et al. Large-scale eradication of rabies using recombinant vaccinia-rabies vaccine. Nature 1991; 354: 520-22. 5. Cooney EL, Collier AC, Greenberg PD, et al. Safety and immunological response to a recombinant vaccinia virus vaccine expressing HIV envelope glycoprotein. Lancet 1991; 337: 567-72. 6. Taylor J, Weinberg R, Lanquet B, et al. Recombinant fowlpox virus inducing protective immunity in non-avian species. Vaccine 1988; 6: 497-503. 7. Taylor J, Weinberg R, Kawaoka Y, et al. Protective immunity against avian influenza induced by a fowlpox virus recombinant. Vaccine 1988; 6: 504-08. 8. Taylor J, Paoletti E. Fowlpox virus as a vector in non-avian species. Vaccine 1988; 6: 466-68. 9. Taylor J, Weinberg R, et al. Non-replicating viral vectors as potential vaccines: recombinant canarypox virus expressing measles virus fusion (F) and hemagglutinin (HA) glycoproteins. Virology 1992; 187: 321-28. 10. Wild F, Giraudon P, Spehner D, et al. Fowlpox virus recombinant encoding the measles virus fusion protein: protection of mice against fatal measles encephalitis. Vaccine 1990; 8: 441-42.
11. Baxby D, Paoletti E. Potential use of non-replicating vectors as recombinant vaccines. Vaccine 1992; 10: 8-9. 12. Taylor J, Trimarchi C, Weinberg R, et al. Efficacy studies on a canarypox-rabies recombinant virus. Vaccine 1991; 9: 190-93. 13. Wiktor TJ, Macfarlan RI, Reagan KJ, et al. Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene. Proc Natl Acad Sci USA
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Kieny MP, Lathe R, Drillien R, glycoprotein from a recombinant
al. Expression of rabies virus vaccinia virus. Nature 1984; 312:
et
163-66. 15. Smith JS,
Yager PA. A rapid tissue culture test for determining rabies neutralizing antibody. In: Kaplan MM, Koprowski H. Laboratory techniques in rabies. Geneva: WHO Monogr Series, 1973: 23. 16. Piccini A, Perkus ME, Paoletti E. Vaccinia virus as an expression vector. Methods Enzymol 1987; 153: 545-63. 17. Goebel SJ, Johnson GP, Perkus ME, et al. The complete DNA sequence of vaccinia virus. Virology 1990; 179: 247-66. 18. Goebel SJ, Johnson GP, Perkus ME, et al. Appendix to "The complete DNA sequence of vaccinia virus". Virology 1990; 179: 517-63. 19. Klietmann W, Schottle A, Kleitmann B, et al. A large scale antirabies immunization study in humans using HDCS vaccine: prophylactic vaccination using different routes of application and post-exposure treatment combined with and without simultaneous serum administration. In: Kuwert E, Wiktor T, Koprowski H, eds. Cell culture rabies vaccines and their protection effect in man. Geneva: International Green Cross, 1981: 330-37. 20. Kuwert EK, Barsenbach C, Wemer J, et al. Early/high and late/low responders among HDCS vaccinees. In: Kuwert EK, Wiktor T, Koprowski H, eds. Cell culture rabies vaccines and their protection effect in man. Geneva: International Green Cross, 1981: 160-67. 21. Etlinger HM, Altenberger W. Overcoming inhibition of antibody responses to a malaria recombinant vaccinia virus caused by prior exposure to wild type virus. Vaccine 1991; 9: 470-72.
Double-blind crossover comparison of human and porcine insulins in patients reporting lack of
hypoglycaemia awareness
There has been much debate about reports that insulin-treated diabetic patients lose of hypoglycaemic symptoms on awareness changing from porcine to human insulin. In a
some
double-blind,
crossover
study,
we
sought
differences between porcine and human insulin in the frequency and characteristics of hypoglycaemic episodes among patients who reported a reduction of awareness of hypoglycaemia after changing treatment.
We studied 50 patients referred by their physicians because of complaints of lack of awareness of hypoglycaemia on human insulin. They had had diabetes for a mean of 20 (SD 12) years and 70% had good or acceptable glycaemic control. Each patient was treated in a double-blind manner for four 1-month periods, two with human and two with porcine insulin, in random order. Only 2 patients correctly identified the sequence of insulin treatments used; 8 or 9 would have been expected to do so by chance alone. The mean percentage of hypoglycaemic episodes associated with reduced or absent awareness was 64% (SD 30%) for human insulin and 69% (31%) for porcine insulin.
We could find no statistically significant differences between the insulin species with respect to glycaemic control or the frequency, timing, severity, or awareness of hypoglycaemia. Reduced hypoglycaemia awareness is common with both human and porcine insulins.
Introduction In 1987 Berger and colleagues! reported a reduction in hunger and sweating in a group of patients with insulindependent diabetes after a change in treatment from porcine to human insulin. This report led to speculation that hypoglycaemia induced by human insulin was associated with less sympatheticoadrenal activation than that induced by animal insulins. Subsequent conflicting findings on the
ADDRESSES: Department of Endocrinology, Diabetes, and Metabolism, Prince of Wales Hospital, Randwick (S. Colagiuri, FRACP); Novo Nordisk Pharmaceuticals, North Rocks (J J. Miller, PhD); and School of Mathematical Sciences, University of Technology, Sydney (P. Petocz, PhD), New South Wales, Australia. Correspondence to Dr Stephen Colagiuri, Department of Endocrinology, Diabetes, and Metabolism, Prince of Wales Hospital, High Street, Randwick, New South Wales 2031, Australia.
