Experimental anthrax vaccines: efficacy of adjuvants combined with protective antigen against an aerosol Bacillus anthracis spore challenge in guinea pigs

Vaccine, Vol. 13, No. 18, pp. 1779-17134, 1995 Elsevier Science Ltd Printed in Great Britain

0264-410x(95)00139-5

Experimental anthrax vaccines: efficacy of adjuvants combined with protective antigen against an aerosol BaciZZus anthracis spore challenge in guinea pigs Bruce Ivins*, Patricia Fellows*, Louise Pitt?, James Estepj-, Joseph Farchaus *, Arthur Friedlander* and Paul Gibbs$ The eficacy of several human anthrax vaccine candidates comprised of d@erent adjuvants together with Bacillus anthracis protective antigen (PA) was evaluated in guinea pigs challenged by an aerosol of virulent B. anthracis spores. The most eficacious vaccines tested were formulated with PA plus monophosphoryl lipid A (MPL) in a squalenel IecithinlTween 80 emulsion (SLT) and PA plus the saponin QS-21. The PA+MPL in SLT vaccine, which was lyophilized and then reconstituted before use, demonstrated strong protective immunogenicity, even after storage for 2 years at 4°C. The MPL component was required for maximum eficacy of the vaccine. Eliminating lyophilization of the vaccine did not diminish its protective ejhcacy. No signtjkant alteration in eficacy was observed when PA was dialyzed against d@erent bufiers before preparation of vaccine. PA+MPL in SLTproved superior in eficacy to the licensed United States human anthrax vaccine in the guinea pig model. Keywords: Anthrax;

Bacillus mfhracis; vaccine

efficacy

During the past several years, there has been considerable research devoted to the development and testing of new, experimental human anthrax vaccines’-‘. Recently, some of these studies have focused on evaluating various adjuvants combined with purified Bacillus anthracis protective antigen (PA)‘.7. The human anthrax vaccines currently licensed in the United Kingdom and the United States have alum and aluminum hydroxide, respectively, as adjuvants. With the United States vaccine, local reactogenicity has been reported in about one-third of vaccinees”, and a total of six immunizations within 18 months are required, followed by yearly Thus, although these human boosters thereafter”. anthrax vaccines are immunogenic in both humans and experimental animals’-7*‘0~“, there is none the less a strong research effort to develop a new, better characterized vaccine with lower reactogenicity, improved efficacy and a reduced immunization schedule. *Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA. tApplied Research Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702501 1, USA. IBiometrics and Information Management Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702501 1, USA. $To whom all correspondence should be addressed. (Received 29 March 1995; revised 5 July 1995; accepted 5 July 1995)

In recent years, several new adjuvants for potential human use have been described’6’9. Among these adjuvants are monophosphoryl lipid A (MPL)20,21, threon$yuramyl dipeptide (T-MDP)22.23, the saponin QS-21 1 , muramyl tripeptide covalently linked to dipalmitoyl phosphatidylethanolamine (MTP-PE)25p27, and several stable, microfluidized oil-in-water emulsions actin as antigen vehicles16*27,28.In research reported in 1992B we studied the efficacy of various adjuvants combined with PA against intramuscular spore challenge in guinea pigs. As we are interested in protecting against inhalation anthrax as well as gastrointestinal and cutaneous anthrax, here we examined vaccine efficacy against an aerosol challenge of virulent B. anthracis Ames strain spores. The ultimate objective of our investigations is to develop a human anthrax vaccine that is safe, efficacious against all known forms of anthrax, less reactogenic than the current vaccines, and that requires a minimum number of doses to elicit high-level, long-term immunity.

MATERIALS

AND METHODS

Animals

Female Hartley guinea pigs, 356400 g, were obtained from Charles River Laboratories, Wilmington, MA. The animals were immunized intramuscularly (i.m.) at 0 and 4 weeks, then were challenged at 10 weeks with an

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Experimental anthrax vaccines: B. lvins et al aerosol of spores of the B. anthrucis Ames strain. Two days before challenge, the animals were anesthetized and bled by cardiac puncture. Sera were assayed for antibody to PA by enzyme-linked immunosorbent assay (ELISA) as described previously”. Vaccines Anthrax Vaccine Adsorbed (MDPH-A VA). The anthrax vaccine licensed for human use in the United States consists of aluminum hydroxide-adsorbed supernatant material, primarily PA, from fermentor cultures of a toxinogenic, nonencapsulated strain of B. anthracis, V770-NPl-R”. The concentration of PA in MDPHAVA is not defined. PA was produced in fermentor cultures of B. anthracis ASterne_l(pPAl02)CR4, a recombinant, nonsporulating strain (pXO1 ~, pXO2-, pPA102+) that does not synthesize capsule, lethal factor or edema factor. PA was purified by anion exchange high pressure liquid chromatography, and the preparations were stored at - 70°C in buffer containing 25 mM diethanolamine, 50 mM NaCl, 2 mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride, pH 8.9. PA was dialyzed for 24 h at 4°C against selected buffers before addition to the appropriate adjuvant. Each dose of vaccine contained 50 ,ug of PA in a total volume of 0.5 ml. The PA was combined with adjuvants as follows: Aluminum hydroxide (Alhydrogel). PA (1200 ,ug) in 0.4 ml phosphate-buffered saline (PBS)“, pH 7.4, was added to 2.4 ml of Alhydrogel (1.3% Al,O,, equivalent to 2.0% AI(O and 9.2 ml of PBS. Each 0.5-ml dose contained 0.7 mg of metallic aluminum. QS-21. Three milliliters of 10 mM sodium phosphate, pH 6.0, was added to 6 mg of QS-21, then 0.6 ml of the solution was added to 0.4 ml (1200 pug) of PA and 11 ml of PBS. Each dose contained 50 pug QS-21. MF59 emulsion. This microfluidized emulsion contained 0.5% (v/v) Tween SO, 0.5% (v/v) Span 85 and 5% (v/v) squalene in water and was mixed 1:l (v/v) with PA (200 ,ug ml ~ ‘) in PBS. MF59 emulsion+MTP-PE. Muramyl tripeptide (Nacetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine) covalently bound to dipalmitoyl phosphatidylethanolamine was added to the MF59 emulsion above. The final MTP-PE dose after mixing 1:l (v/v) with PA (200 pug ml ‘) in PBS was 50 pg. SAF-M emulsion. The SAF-M microfluidized emulsion consisted of 10% squalane, 0.4% Tween 80 and 5.0% Pluronic block copolymer L121 in PBS. It was mixed 1: 1 (v/v) with antigen (200 pug ml - ‘) in PBS. SAF-M emulsion-t T-MDP. Threonyl muramyl dipeptide (N-acetylmuramyl-L-threonyl-D-isoglutamine) was added to the SAF-M emulsion above. The final T-MDP dose after mixing with an equal volume of PA (200 pug ml ~ ‘) in PBS was 50 pg. SqualenellecithinlTween 80 emulsion (SLT) +MPL. Each 0.5-ml dose of this vaccine contained 50 ,ug PA,

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2% (v/v) squalene, 0.24% (w/v) lecithin, 0.08% (v/v) Tween 80 and either 50 or 100 pug of MPL. Eighty-dose quantities of the vaccine were prepared by first dissolving either 5 or 10 mg of MPL in 1.0 ml of squalene containing 12% (w/v) lecithin. Four milligrams of PA in buffer was then mixed with 0.8 ml of MPL/squalene/ lecithin by using a Potter-Elvehjem grinding tube. Twenty milliliters of 0.16% (v/v) Tween 80 in water was added to the PA/MPL/squalene/lecithin, and the final volume was then brought up to 40 ml with water. After thoroughly mixing, the PA+MPL in SLT was dispensed into lo- or 20-ml vaccine vials, lyophilized, stoppered and stored at 4°C. Immediately before immunization the vaccine was reconstituted to an emulsion with PBS. In one experiment, PA was dialyzed into various buffers before the addition of MPL in SLT and lyophilization. The buffers were all adjusted to pH 7.65 and included: PBS; 5 mM HEPES, 50 mM NaCl; 5 mM glycyl glycine, 50 mM NaCl; 5 mM Tris, 50 mM NaCl; and 5 mM sodium citrate, 50 mM NaCl. In the final experiment, one vaccine preparation was not lyophilized, whereas another lyophilized preparation lacked MPL. The source of Alhydrogel was Superfos Biosector a/s, Denmark, distributed by E.M. Sargeant Pulp and Chemical Co., Inc., Clifton, NJ. MPL was purchased from Ribi Immunochem Research, Inc., Hamilton, MT. The saponin QS-21 was the gift of Dr Oscar Kenshala, Cambridge Biotech, Worcester, MA. MF59, MTP-PE, SAF-M and T-MDP were the gifts of Dr Gary Van Nest. Chiron Corporation, Emeryville, CA. MDPHAVA (lot FAV 008) was obtained from the Michigan Department of Public Health, Lansing, MI. Squalene, lecithin, and Tween 80 were obtained from the Sigma Chemical Company, St. Louis, MO. Spore challenge The virulent Ames strain of B. anthracis, obtained from the US Department of Agriculture, Ames, IA, was cultured with shaking in Leighton-Doi mediumx2 for 4 days at 30°C. Spores were harvested by centrifugation and washed in sterile distilled water as described previously6, then purified by centrifugation through 58% Renografin-76. The spores were washed again, resuspended in 1% phenol and stored at 4°C. For aerosol challenge, spores were suspended to a concentration of ca 1 x lo9 c.f.u. ml-‘, then were heat-shocked at 60°C for 45 min. In groups of 12, guinea pigs received an aerosol challenge of the spores by a nose-only exposure system contained within a Class III biological safety cabinet. This rodent-exposure system is a Plexiglas tower that can hold 12 guinea pigs. Animals were restrained in holders that did not restrict their respiratory function, and the holders were attached to the tower. Only the tips of the guinea pigs’ noses were exposed to the aerosol. The aerosols were generated by a three-jet Collison nebulize?’ containing 8 ml of the spores at the required dilution in sterile water. This nebulizer, driven by compressed air at 26 lb in ~ 2, produced an aerosol at a flow rate of 7.5 1 min- ’ and a mass median aerosol diameter of 1.2 pm. This aerosol was mixed with 4.5 1 min- ’ of secondary air for a total system flow rate of 12 1 min _ ’

Experimental anthrax vaccines: B. bins et al. Table 1

Protective

efficacy of various adjuvants

combined

with PA against an aerosol challenge of virulent B. anfhracis sporesa

Vaccine

Survived/total

(W

Anti-PA titers’

TTD” (days)

First aerosol challenge triap PA + MPL in SLT e,f.g MDPH-AVAe,g PA in MF59” PA+MTP-PE in MF5ge PA+Alhydrogele PBS

9/l 8 5/l 9 3118 2/l 9 1I20 0119

(501

11 220 9941 18 836 14 962 7499 Cl0

17.31 6.91 5.79 6.33 5.45 2.38

Second aerosol challenge PA+QS-21 e,g PA in SAF-M’ MDPH-AVAe PA+T-MDP in SAF-Me,’ PBS

8180 4120 4120 l/l5 o/17

8035 11 350 7.45 12 023
11.86 6.69

1;;’ (0)

triar (40) (20) (20)1758

7.14 2.17

“Female Hartley guinea pigs, 350-400 g, were immunized i.m. at 0 and 4 weeks with 0.5 ml of the indicated vaccine, then aerosol challenged at 10 weeks with B. anthracis Ames strain spore. Doses of PA, MPL, MTP-PE, T-MDP and QS-21 were 50 ,ug. *Reciprocal geometric mean anti-PA serum ELISA titers 2 days before challenge. 7TD=Harmonic mean time to death. dGuinea pigs received a single aerosol challenge dose of ca 216 LD,,. eBy Log-Rank test, survival distribution function significantly different from that of PBS (P
before traveling along a mixing tube and entering into the exposure system at the inlet aerosol chamber. Here the aerosol divided, with 6 1 mini ’ flowing into the delivery channel to the nose ports where it was distributed evenly through metering orifices. The other 6 1 min- ’ was sampled using an all glass impinger (AGI)34. The AGI contained a critical orifice to regulate flow at 6 1min ‘. This sampler required a sustained vacuum of at least 15’ of Hg to maintain the critical pressure ratio across the orifice, which, in turn, produced sonic velocity. The spores were impinged in 10 ml of sterile water contained in the sampler. The aerosols were sampled continuously throughout each entire lo-min exposure trial. Dilutions of the sampled aerosol were plated onto Tryptic Soy Agar (Difco, Detroit, MI) and colonies counted after 16 h of incubation at 37°C. The aerosol concentrations were then calculated, and the inhaled dose (c.f.u. guinea pig- ‘) estimated by using Guyton’s formula for minute volume calculations of the rodentss5. The spore dose actually retained in the lungs as well as the number of spores ingested (either during aerosolization, or as a result of ciliary clearance of the spores from the respiratory tract) was not determined. One median lethal dose (LD,,) was equivalent to 7.9 x lo4 inhaled spores (L. Pitt, unpublished observations). Animals were monitored daily for 3 weeks for death or survival. Harmonic mean times to death (TTD) were calculated for each immunization group. Differences in final mortality were compared by using Fisher’s exact test. The more sensitive product-limit method (Lifetest Procedure, SAS, Cary, NC), which takes into account the rate of death, was used to estimate the survival distribution function?, and differences between survival distribution functions were tested by the Log-Rank statistic.

RESULTS Efficacy of PA combined with different adjuvants

The protective efficacy of the current U.S. human anthrax vaccine, MDPH-AVA, was compared to PA combined with Alhydrogel, SAF-M, SAF-M+T-MDP, MF59, MF59+MTP-PE, QS-21 and MPL (50 pg per

dose) in SLT. The data from the first two aerosol challenge experiments (Table 1) demonstrate the relative efficacy of the various vaccines. By Fisher’s exact test only PA+MPL in SLT, MDPH-AVA (first aerosol challenge trial), and PA+QS-21 were significantly more protective against mortality than PBS (P10.05). Comparison of survival distribution functions by the LogRank statistic indicated that all the vaccines were significantly better than PBS (P
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Experimental anthrax vaccines: B. bins et al. Table 2 Immunological years at 4°C”

stability

of PA+MPL

in SLT stored for 2

Vaccineb

Survived/ total

(“A)

Anti-PA titers=

TTD’ (days)

PA+MPL in SLTe,’ PBS control

13119 o/4

(68) (0)

17 782
20.05 3.20

“Female Hartley guinea pigs, 350-400 g, were immunized i.m. at 0 and 4 weeks with PA (50 pg)+MPL (100 pug) in SLT, then aerosol challenged at 10 weeks with ca 83 LD,, 6. anfhracis Ames strain spores. bPA+MPL in SLT was prepared, lyophilized and stored for 2 years at 4°C. Immediately before use the vaccine was reconstituted in PBS. %eciprocal geometric mean anti-PA serum ELISA titers 2 days before challenge. ‘TTD=Harmonic mean time to death. eBy Log-Rank test, survival all distribution function significantly different from that of PBS (P
Table 3 Effect of various buffers on efficacy of vaccines containing PA+MPL in SLTa Survived/ total

Bufferb SH;di;msc/tratee.’ Tris? PBS,‘ Glycyl glycine’,’ PBS alone (control)

16116 16116 14116 10112 12116 014

(“A) (100) I::;) (83) I::’

Anti-PA titersC

TTDd (days)

31 622 25 482 27 384 29 427 23 713
130.72 41.14 22.60 2.40

“Female Hartley guinea pigs, 350-400 g, were immunized i.m. at 0 and 4 weeks with PA (50 pg)+MPL (100 pg) in SLT, then aerosol challenged at 10 weeks with ca 35 LD,, 6. anthracis Ames strain spores. bAll buffers were adjusted to pH 7.65. Except for PBS, the buffers contained 5 mM of the indicated compound and 50 mM of NaCI. “Reciprocal geometric mean anti-PA serum ELISA titers 2 days before challenge. dTTD=Harmonic mean time to death. eBy Log-Rank test, survival distribution function significantly different from that of PBS (f
Table 4

Influence of MPL and lyophilization

Vaccine PA+MPL in SLTd,e (lyophilized) PA+MPL in SLTae (unlyophilized) PA (without MPL) in SLTd (lyophilized) PBS control

on vaccine efficacya

Survived/ total

Anti-PA titersb

TTD”

(W

16119

(84)

28 013

56.35

17120

(85)

19 952

81.17

8120 o/4

(40) (0)

16 237
10.01 2.67

(Days)

“Female Hartley guinea pigs, 350-400 g, were immunized i.m. at 0 and 4 weeks with PA (50 pg) in SLTkMPL (100 pg), then aerosol challenged at 10 weeks with ca 145 LD,, B. anfhracis Ames strain spores. bReciprocal geometric mean anti-PA serum ELISA titers 2 days before challenge. “TTD=Harmonic mean time to death. By Log-Rank test, survival distribution function significantly different from PBS (f
PA+MPL in SLT, lyophilized, then reconstituted immediately before immunization; PA+MPL in SIT, made immediately prior to use and not lyophilized; and PA without MPL in SLT, lyophilized, then reconstituted immediately before use.

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We wanted to know if the efficacy of PA+MPL in SLT was due to the presence of MPL or to the fact that the vaccine was lyophilized, thus forcing contact between antigen and oil. Both the lyophilized and unlyophilized emulsions containing MPL and PA protected well against an aerosol spore challenge (Table 3). Eliminating MPL from the emulsion decreased protective efficacy significantly (P
DISCUSSION Guinea pigs are used frequently in anthrax vaccine efficacy studies3,5. They are quite susceptible to both parenteral and aerosol B. anthracis spore challenge ‘,2.5.13.29. Numerous studies have shown that they are difficult to rotect by immunization with human _ anthrax vaccines P.3~5,7,‘7,29 . Our data clearly indicate that new, potential human-use adjutants can be combined vaccines with B. anthracis PA to generate candidate that protect guinea pigs against aerosol spore challenge. Alhydrogel was the only adjuvant used in these studies that is approved by the US Food and Drug Administration. When combined with PA (Table 1), it did not significantly protect guinea pigs against mortality, whereas the licensed US human anthrax vaccine, MDPH-AVA, which contains Alhydrogel as an adjuvant, protected 126% of the guinea pigs from aerosol spore challenge. PA combined with either the SAF-M or the MF59 emulsions provided little protection against mortality, even after addition of T-MDP or MDP-PE. QS-21+PA significantly protected the guinea pigs from death. PA+MPL in SLT provided strong protection against aerosol challenge, especially with an MPL dose of 100 pg. This vaccine was also efficacious against an i.m. spore challenge (B. Ivins, unpublished observations). In these studies, we demonstrated that: lyophilization of PA+MPL in SLT did not affect protective immunogenicity; MPL was an important component of the vaccine; the lyophilized vaccine was stable for at least 2 years at 4°C; and varying the buffer against which PA was is dialyzed did not significantly affect the vaccine’s protective efficacy. (We currently use 5 mM sodium citrate, 50 mM NaCl, pH 7.65 as the buffer.) All the vaccines tested significantly protected guinea pigs by extending the time to death (thus decreasing the group rate of mortality) compared to PBS controls. The high level of protection generated by PA+MPL in SLT may have been due in part to the fact that MPL, a potent stimulator of cell-mediated immunity (CMI), is dissolved in the oil droplets of the emulsion. Association of hydrophobic regions of the PA molecule with oil droplets containing MPL may faciliate both interaction with antigen-presenting cells (APC) and stimulation of specific immune system activities such as B-cell proliferation, macrophage activation and augmentation of specific antibody production’6*20,2’. The increased anti-PA antibody titer and the enhanced protection observed with this vaccine in Tables 2 and 3 and 4, in comparison to that seen in Table 1, may be due to the increased dose of MPL (100 pug). The other adjuvants

Experimental anthrax vaccines: B. bins et al.

used in these studies also exert their immunomodulatory effects by acting on the various components of the immune system. Muramyl dipeptide and its analogues, T-MDP and MTP-PE, are adjuvants that promote CM1 and production of antibodies of specific protective isotypes22,23. QS-21 stimulates IgG responses and CMI, especially through Class I major histocompatibilityrestricted cytotoxic lymphocytes14,24. Aluminum salts and mineral oil emulsions strongly enhance humoral but not CM1 responses’6,‘7. Emulsions such as MF59 and SAF-M may help target antigens to specific APC’5*‘6. We and others previously suggested that humoral immune responses were not solely responsible for protection against anthrax’~‘~“’ and our data reinforce that hypothesis. Indeed, in this as in other studies1,235,7,29,38, there was no strict correlation between anti-PA ELISA titers and survival. Polyclonal titers may reflect the antibody response to many regions of the PA molecule but not necessarily to the specific protective epitopes. Indeed, in vaccines containing aluminum salts, antigen and adjuvant interact strongly through electrostatic forces39*40.When PA is bound to Alhydrogel in PBS at pH 7.4, the PA possesses a net negative charge, and the Alhydrogel has a net positive charge. Preferential binding of certain negatively charged regions of the PA may obscure certain protective epitopes from APC. Interaction of PA with oil droplets in an emulsion such as SLT, however, may involve hydrophobic rather than highly charged regions of the molecule, possibly exposing certain critical sites to the immune response. Although antibodies certainly play a role in specific immunity to anthrax1’,4’-43, they do not appear to be entirely responsible for that specific immunity. Indeed, CM1 responses as mediated through activated macrophages may be critical. In inhalation anthrax, macrophages transport the B. anthracis spores from the alveoli to the regional lymph nodes”. Furthermore, macrophages are the principal target for anthrax lethal toxin44. Clearly, any specific immune alteration of macrophage competence in the direction of increased ability to kill B. anthrucis cells or to resist lethal toxin action could be of substantial benefit to the host. In earlier studies’ we noted that various adjuvants combined with PA protected guinea pigs against an i.m. challenge of B. anthracis spores. The data presented here extend those findings into the area of aerosol spore challenge and thus have relevance to the development of specific immunity to inhalation anthrax. Our next step will be to investigate the most promising experimental anthrax vaccine candidates, including PA+MPL in SLT and PA+QS-21, with respect to efficacy in other animal species and duration of protection following immunization. All of these studies are critical prerequisites to making a rational selection of a new human anthrax vaccine. Advances in adjuvant technology as well as in antigen production and purification methods will contribute significantly to vaccine development.

for excellent technical assistance, Catherine Wilhelmsen for animal necropsy, and Susan Welkos, George Anderson, Katheryn Kenyon and Steve Little for reviewing this manuscript. In conducting the research described in this report, the investigators adhered to the tiide for the Care and Use of Laboratory Animals, as promulgated by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council. The facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care. The views of the authors do not purport to reflect the positions of the Department of the Army or the Department of Defense. REFERENCES 1

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ACKNOWLEDGEMENTS The authors are grateful to Dr J.T. Ulrich for suggestions regarding the formulation of the PA+MPL in emulsion. We also thank Wil Ribot and Scott Jendrek

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Turnbull, P.C.B., Quinn, C.P., Hewson, R., Stockbridge, MC. and Melling, J. Protection conferred by microbiallysupplemented UK and purified PA vaccines. Proceedings of the International Workshop on Anthrax, 11-13 April 1989, Winchester, UK. SalisburyMed. Bull. 1990, 66 (Special Suppl.), 89-9 1 Turnbull, P.C.B., Leppla, S.H., Broster, M.G. and Melling, J. Antibodies to anthrax toxin in humans and guinea pigs and their relevance to protective antigen. Med. Microbial. Immunol. 1988, 177,293-303 Turnbull, P.C.B. Anthrax vaccines: past, present and future. Vaccine 1991, 9, 533-539 Hambleton, P. and Turnbull, P.C.B. Anthrax vaccine development: a continuing story. In: Bacteria/ Vaccines (Ed. Mizrahi, A.). Advances in Biotechnological Processes, vol. 13. Alan R. Liss, New York, 1990, pp. 105-122 Ivins, B.E. and Welkos, S.L. Recent advances in the development of an improved human anthrax vaccine. Eur. J. Epidemiol. 1988, 4, 12-l 9 Ivins, B.E., Welkos, S.L., Knudson, G.B. and Little, S.F. Immunization against anthrax with aromatic compound-dependent (Aro-) mutants of Bacillus anthracis and with recombinant strains of Bacillus subfilis that produce anthrax protective antigen. Infect. Immun. 1990, 56, 303-308 Ivins, B.E., Welkos, S.L., Little, SF., Crumrine, M.H. and Nelson, G.O. Immunization against anthrax with Bacillus anfhracis protective antigen combined with adjuvants. Infect. Immun. 1992, 60, 662-668 Welkos, S.L. and Friedlander, A.M. Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice. Microb. Pathog. 1988, 5, 127-139 Welkos, S., Becker, D., Friedlander, A. and Trotter, Ft. Pathogenesis and host resistance to Bacillus anfhracis: a mouse model. Proceedings of the International Workshop on Anthrax, 11-13 April 1989, Winchester, UK. Saksbury Med. Bull. 1990, 68 (Special Suppl.), 49-52 Brachman, P.S., Gold, H., Plotkin, S.A., Fekety, F.R., Werrin, M. and Ingraham, N.R. Field evaluation of a human anthrax vaccine. Am. J. Pub/. H/N, 1962, 52, 632-645 Brachman, P.S. and Friedlander, A.M. Anthrax. In: Vaccines (Eds Plotkin, S.A. and Mortimer, Jr, E.A.). W.B. Saunders Co., Philadelphia, 1994, pp. 729-739 Friedlander, A.M., Welkos, S.L., Pitt, M.L.M. et a/. Postexposure prophylaxis against experimental inhalation of anthrax. J. Infect. Dis. 1993, 167, 1239-1242 Ivins, B.E., Fellows, P.F. and Nelson, G.O. Efficacy of a standard human anthrax vaccine against Bacillus anfhracis spore challenge in guinea pigs. Vaccine 1994, l-2, 872-874 Kensil, C.R., Patel, U., Lennick, M. and Marciani, D. Separation and characterization of saponins with adjuvant activity from Quillaja saponaria molina cortex. J. lmmunol. 1991, 146, 431437 Woodard, L.F. Surface chemistry and classification of vaccine adjuvants and vehicles. In: Bacterial Vaccines (Ed. Mizrahi, A.). Advances in Biotechnological Processes, vol. 13. Alan R. Liss, New York, 1990, pp. 281-306

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Experimental anthrax vaccines: B. bins et al. Allison, A.C. and Byars, N.E. Immunological adjuvants and their mode of action. Biotechnology 1992, 20, 431-449 17 Edelman, Ft. and Tacket, C.O. Adjuvants. Int. Rev. Immunol. 1990, 7, 51-66 18 Myers, K.R. and Gustafson, G.L. Adjuvants for human vaccine usage: a rational design. In: Vaccines and lmmunofherapy (Ed. Cryz, Jr S.J.). Pergamon Press, New York, 1991, pp. 404-411 19 Sandhu, J.S. Engineered human vaccines. Cm. Rev Biotechno/. 1994, 14, l-27 20 Ribi, E., Cantrell, J. and Yakayama, K. A new immunomodulator with potential clinical applications: monophosphoryl lipid A, a detoxified endotoxin. Immunol. News/. 1985, 6, 33-36 21 Ribi, E., Ulrich, J.T. and Masihi, K.N. lmmunopotentiating activities of monophosphoryl lipid A. In: lmmunopharmacology of fnfectious Disease. Vaccine Adjuvants and Modulators of Non-Specific Resistance (Ed. Majde, J.A.). Progress in Leukocyte Biology, vol. 6. Alan R. Liss, New York, 1987, pp. 101-112 22 Allison, A.C. and Byars, N.E. An adjuvant formulation that selectively elicits the formation of antibodies of protective isotypes and of cell mediated immunity. J. Immunol. Meth. 1986, 95, 157-l 68 23 Byars, N.E. and Allison, A.C. Adjuvant formulation for use in vaccines to elicit both cell-mediated and humoral immunity. Vaccine 1987, 5, 223-228 24 Kensil, C.R., Newman, M.J., Coughlin, R.T. et al. The use of Stimulon adjuvant to boost vaccine response. Vaccine Res. 1993, 2, 273-281 25 Wintsch, J., Chaignat, CL., Braun, D.G. et al. Safety and immunogenicity of a genetically engineered human immunodeficiency virus vaccine. J. Infect. &. 1991, 163, 219-225 26 Keitel, W., Couch, R., Bond, N., Adair, S., Van Nest, G.A. and Dekker, C. Pilot evaluation of influenza virus vaccine (IVV) combined with adjuvant. Vaccine 1993, 11, 909-913 27 Valensi, J.P., Carlson, J.R. and Van Nest. G.A. Svstemic cytokine profiles in BALB/c mice immunized with trivalent influenza vaccine containing MF59 oil emulsion and other advanced adjuvants. J. Immunol. 1994, 153, 40294039 28 Haigwood, N.L., Nara, P.L., Brooks, E. et al. Native but not denatured recombinant human immunodeficiency virus type 1 gp120 generates broad-spectrum neutralizing antibodies in baboons. J. Viral. 1992, 66, 172-182 29 Little, S.F. and Knudson, G.B. Comparative efficacy of Bacillus anthracis live spore vaccine and protective antigen vaccine against anthrax in the guinea pig. Infect. fmmun. 1986, 52, 509-512 16

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