A comparison of antibody responses to veterinary vaccine antigens potentiated by different adjuvants

Vaccine, Vol. 15. No. 17/l& pp. 1902-1907, 1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X197 $17+0.00

PII: SO264-410X(97)00136-9 ELSEVIER

A comparison of antibody responses to veterinary vaccine antigens potentiated by different adjuvants William R. Usinger Six adjuvant formulations were compared for their ability to potentiate the primary and memory antibody responses in mice to three companion animal vaccine immunogens-feline leukemia virus (FeLV), f e 1ine immunodeficiency virus (FIV), and a recombinantly-derived heartworm antigen. The combination of a novel bacterial immunostimulatol; gliding bacterial adjuvant (CBA), either adsorbed onto an aluminum hydroxide gel (RehydragelTM HPA), or emulsified with a vehicle of polyalcohol and detergent, elicited the strongest memory responses to both virus preparations. Both forms of aluminum hydroxide gels administered without GBA gave similar levels of adjuvant effects, on par with or reater than those generated by incomplete Freund’s 74 adjuvant (IFA). The Acemannan immunostimulant was not effective in increasing the responses to the virus antigens, but increased the primary response to the heartworm antigen over tenfold from control levels. All preparations appeared to be well tolerated, with no detectable adverse reactions observed in any of the 250 mice used. The proven safety of aluminum hydroxide adjuvants and the apparent absence of adverse reactions seen with CBA make this vehicle~adjuvant formulation worthy of additional study. 0 1997 Elsevier Science Ltd. Keywords: adjuvant; FeLV, feline leukemia Freund’s adjuvant; vaccine; vehicle

virus;

FIV, feline

immunodeficiency

More than one billion doses of various vaccines complexed with aluminum based adjuvants have been administered to humans since the 1920s when it was discovered that diphtheria toxoid (DT), partially purified by precipitation with anions and aluminum, was more immunogenic than the soluble toxoid administered alone’. A vast record of acceptable safety and demonstrated efficacy in potentiating protective antibody responses to a broad variety of antigens has been documented over the last 50 years. Owing to the frequent and often severe adverse reactions observed with Freund’s complete adjuvant (FCA), many developers of new adjuvants have chosen aluminum adjuvants as the consensus gold standard against which to compare their new products. Until these new products demonstrate convincing safety and efficacy, aluminum adjuvants (aluminum hydroxide and aluminum phosphate) remain the ‘non-toxic’ alternative to FCA and as such are the only adjuvants approved and licensed for use with human vaccines in the US.

Immusine, Inc., 25 Via Floreado, Orinda, CA 94563, USA. Tel.: (510) 254-7137; fax: (510) 254-9047. (Received 19 February 1997; revised version received 29 April 1997; accepted 5 May 1997)

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virus:

GBA, gliding

bacterial

adjuvant:

IFA. incomplete

There are several significant limitations to the use of aluminum adjuvants that have spawned the search for new adjuvants. Perhaps foremost is aluminum’s inability to augment cellular immune responses thought to be required for protection to many diseases, including viruses like HIV and herpes simplex, and intracellular parasites’. In addition, there is the perception that modern subunit and peptide-based vaccines, themselves generally not strong immunogens, will require more potentiation than can be achieved with aluminum alone. Thus, in some vaccine development aluminum-based programs, adjuvants are being combined with an immunomodulator. For these reasons, potent and non-toxic adjuvants are of great interest to those developing vaccines. A compendium of over 70 of the better studied adjuvants, excipients and immunomodulatory hormones currently being evaluated has recently been published’. Further, the formulation of effective combinations of vaccine antigens, adjuvants,, interleukin hormones and oils has been an area of intense interest. This notion of combining individually effective immune stimulants has a very well documented basis of effectiveness as exemplified by FCA. The mineral oil component of FCA forms an oil-in-water emulsion with aqueous vaccine and serves as an antigen reservoir or ‘depot’, preventing rapid elimination and promoting sustained

Antibody responses

release of antigen. The killed Mycohacteria in FCA is a potent stimulator of lymphokine release, promoting additional immune responses to those induced by the oil alone (incomplete Freund’s adjuvant, IFA). Guided by the example of Freund’s adjuvant and the inherent logic of formulating biological response modifiers with immunostimulatory vehicles, we demonstrated that the potency of a novel bacterial polysaccharide’, gliding bacteria adjuvant (GBA), could bc similarly enhanced when emulsified with ‘oil”. Concerns over the toxicity of this vehicle have us to search for a new formulation prompted comprised of GBA and a vehicle with a record of safe and effective use. In this study a widely used sterile, low viscosity aluminum hydroxide (Rehydragcl LVrM) was compared with a newly developed aluminum hydroxide gel with a grcatcr adsorptive capacity and smaller particle size (Rchydragel HPATM) for potentiation of primary and in mice to three memory antibody responses companion animal vaccine antigens. A new formulation of GBA combined with Rehydragel HPATM, and two other adjuvants, Acemannan’lM and IFA, wcrc also examined.

MATERIALS

AND METHODS

Adjuvants

(1) PBS: IO mM phosphate in 150 mM NaCI, pH 7.4, (2)

(3) (4)

(5)

(6)

(7)

was used as the control diluent for each vaccine antigen. Reheis RehydragelTM LV aluminum hydroxide gel (LV): a 0.1 ml dose of vaccine consists of 2 /lg antigen adsorbed onto 0.05 mg aluminum as aluminum hydroxide-l part antigen: 25 parts adjuvant. Reheis RehydragelTM HPA aluminum hydroxide gel (HPA): prepared as above for LV. GBAipolyol: purified GBA polysaccharide (5 /cg per dose) was combined with aqueous vaccine antigen and emulsified prior to immunization with an equal volume of the polyalcohol Pluronic L121 (1.25%) final cont.) (BASF, Parsippany, NJ), Twcen “’ SO (0.2% final cont.) (Sigma Chemical Co., St. Louis, MO) and squalane (5% final cont.) (Sigma). HPA+GBA: concentrated GBA was added to the appropriate amount of each vaccine (see below) and then mixed with the corresponding amount of HPA; i.e. 1 part (vaccine+GBA): 25 parts HPA to achieve a final concentration of 5 /cg GBA per dose. Acemannan lM immunostimulant: this commercial adjuvant (Carrington Laboratories, Irving, TX) is composed of long-chain polydispersed p-( 1,4)-linked mannan polymers interspersed with O-acetyl groups. The recommended intraperitoneal (i.p.) dose of 1 mg kg ’ was used; the material was administered with each vaccine within 4 h of reconstitution with sterile diluent. IFA: a I:1 oil-in-water emulsion was formed with IFA (Sigma) and each vaccine preparation.

to vaccines potentiated

by adjuvants:

W.R. Usinger

Vaccine immunogens

(1) Feline leukemia virus (FeLV) (2 /cg per injection). This preparation is a licensed, chcmically-inactivated purified preparation of whole, intact FeLV (BioTrends International, West Sacramento. CA). It is supplied as a I mg ml ’ sterile solution. (2) Feline immunodeficiency virus (FIV) (2 /lg per injection). Prior to use in this study, the preparation, a sucrose density gradient purified, 1000 x concentrated, live virus preparation (Petaluma Biotcchnologies, Columbia, Strain, Advanced MD), was heat-inactivated for I h at 60°C‘. Supplied at 1.09 mg ml ‘. (3) Recombinantly-derived heartworm antigen (rHW) (I Llg per injection). This preparation was a purified 70 kDa protein isolated from an Escherichia coli genetic construct”. Owing to limited supply, I /lg was used to immunize and subsequently restimulate each mouse.

Immunization

schedule

Fourtcen days following the prcbleed and initial immunization, individual adult BALBic mice were bled, serum was collected and diluted I:5 and frozen until further testing. Following a 5 week rest interval, the mice were each reinjected with the initial vaccine in the absence of adjuvant. Ten days later the mice were rebled, serum was collected and diluted I:5 and kept frozen until testing. All immunizations were 100 ktl per i.p. injection, with five to seven mice per test group. Determination

of antibody response

An enzyme immunoassay (EIA) was performed in which 100 111of each immunizing antigen served as the subsequent target for antibody binding. For FeLV and FIV, each test well (Immulon If, Dynatech, Chantilly, of each immtmogen VA) was coated with 5 /ig ml diluted in PBS. For the heartworm antigen, 2 /!g ml ’ was added per well. Following overnight incubation at 4°C wells were washed six times with PBS and then blocked with a solution of PBS containinin 2% bovine 20 (Sigma). serum albumin (BSA) and 0.1% Tween After 4 h of incubation at room temperature, wells were rewashed and then fresh PBS-2% BSA was added to each well and serial fourfold dilutions of each antiserum sample were made. After overnight incubation in the cold, wells were washed and developed using a rabbit anti-mouse Ig linked with alkaline phosphatasc (Sigma). Following a 2 h incubation at room temperature, a 1 mg ml ’ cationic solution of p-nitrophenylphosphate ( I .O M dicthanolamine buffer, pH 9.8, containing 5 mM MgCI) was added. Reactions were terminated by the addition of 4 M NaOH, approximately 112h following addition of substrate. Absorbance was measured at 405 nm. RESULTS Approximately 250 Balbic mice were randomly assigned to an experimental regimen and then bled and tested by EIA for prc-existing antibodies to the test antigen to which they were assigned. Four mice had

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Antibody responses to vaccines potentiated by adjuvants: W.R. Usinger

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Figure 1 Individual primary and secondary antibody responses in BALB/c mice immunized with 2 {rg FeLV with various adjuvants. Beginning with a 15 dilution, serum was serially diluted 1:4 and examined for binding to FeLV by EIA. Mean responses are indicated in bold. The standard error of the mean response is given for each dilution, (a) Saline control: mice were immunized with FeLV in saline, and subsequently reimmunized without adjuvant. (b) Mice were immunized with FeLV in Rehydragel TM HPA adjuvant, and subsequently reimmunized without adjuvant. (c) Mice were immunized with FeLV in RehydragelTM HPAIGBA adjuvant, and subsequently reimmunized without adjuvant

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Antibody responses to vaccines potentiated by adjuvants: W.R. Usinger Table 1 Summary of primary and secondary antibody responses in BALB/c mice to three companion animal vaccine immunogens-feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), and a recombinantly-derived heartworm antigen (rHW). Values represent the number of fourfold dilutions of serum (starting at 15) required to achieve a 0.2 O.D. in the EIA rHW

FIV

FeLV Adjuvant

1”

2*

AC

1

2

A

1

2

A

Saline Rehydragel LV Rehydragel HPA GBA/polyol HPAIGBA Acemannan IFA

1.5 4.0 4.1 6.1 4.4 1.8 1.8

7.3 7.4 7.4 8.5 8.1 6.3 6.2

5.8 3.4 3.3 2.4 3.7 4.5 4.4

1.6 2.8 3.0 4.7 2.6 2.9 2.9

6.6 5.7 5.0 8.6 7.6 6.4 6.6

5 2.9 2 3.9 5 3.5 3.7

3.2 2.1 3.6 2.6 2.2 1.7 4.4

3.4 5.4 5.7 5.7 4.3 2.4 5.1

0.2 3.3 2.1 3.1 2.1 0.7 0.7

“Primary response %econdary response “Secondary response

minus the primary response

slight responses

at a serum dilution of l/l0 (optical density [O.D.] > 0.3, 20 min) and were eliminated from the study. Qualifying mice had responses of O.D. ~0.1, 20 min, at a I:10 serum dilution. Beginning with a starting dilution of 15, 12 serial fourfold dilutions of test serum were evaluated by EIA. To permit a direct comparison of primary and secondary responses, sera from both immunizations were stored frozen until the day of assay. Individual titration curves and their second order polynomial best tit (with standard errors) for representative responses (HPAIGBA, HPA alone, and control) are shown in Figure I(a)-(c). The linear portion of each average titration curve was used to construct a best fit linear regression. Using this equation, the ‘antibody titer’ for each reaction was defined as the reciprocal of that dilution of serum which yielded an absorbance of 0.20. A summary of the average antibody titer for the entire study is shown in Tuhlr I. Values in Tuble I are expressed as the log 4 dilution which gave an O.D. of 0.2 (excluding for simplicity the initial fivefold dilution of serum).

I .1)00.000

0

Values in Table 1 were converted to absolute titers (the reciprocal of the final dilution) and plotted in Figures 2-4. PBS served as diluent for both GBA and the antigen preparations and is the control for this adjuvant study.

DISCUSSION Initially discovered for its ability to potentiate antibody responses in vitro”, more recent studies of GBA have shown it to be a potent stimulator of lymphokine release from cells of mice, cats and humans with little apparent toxicity4.5. When emulsified in the ‘Syntex formulation’7 with pluronic polyol L121, squalane and detergent, GBA’s adjuvant properties were substantially enhanced’. On the basis of these findings and the reports of others showing enhancement of aluminum’s adjuvant effect by addition of immunomodulatorsX9, this study was designed to examine a new formulation of GBA adsorbed to a high capacity aluminum hydroxide gel. These responses were compared with GBA/polyol and widely used aluminum hydroxide gels I0.000.000

I ’ Response

q 2’

Response

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I

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Adjuvants Figure 2 Average primary and secondary FeLV potentiated by six adjuvants

Adjuvants antibody

responses

to

Figure 3 Average primaty and secondary FIV potentiated by six adjuvants

antibody

responses

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Figure 4 Average primary and secondary antibody heartworm antigen potentiated by six adjuvants

responses

to

GBA. Further, IFA, a well studied and relatively nontoxic adjuvant recently evaluated clinically for use with a whole, killed HIV vaccine, and AcemannanTM, a new adjuvant and a polysaccharide like GBA, were also examined. The vaccine immunogens used in this study were chosen for their proven (FeLV) and potential use in important companion animal vaccines. We believe that a new adjuvant formulation with a demonstrated record of safety and effectiveness, in combination with one or more veterinary vaccines, would significantly improve companion animal health and provide a strong base from which to enter the human vaccine arena. Inspection of the composite results (Table I) indicates a number of findings.

without

(1) The

strongest adjuvant was GBA/polyol. It promoted the greatest memory responses to all three antigens and a titer of z 10’ to both FeLV and FIV. (2) The new formulation of RehydragelTM HPA with GBA was nearly as potent as GBA/polyol in promoting memory. It was generally superior to using HPA alone, inducing responses of over two to nearly 37 times greater than those induced by HPA to FeLV and FIV. (3) RehydragelTM HPA and LV were remarkably similar to each other in augmenting both primary and secondary responses. Possible differences in potency of these adjuvants might be more readily observed if advantage was taken of their varying adsorptive capacities. That variable was not tested in this study. (4) Markedly lower in potency was AcemannanTM; it promoted the weakest responses of the adjuvants tested, with memory responses lower than the saline control to all three antigens. A substantial secondary response was induced by the two viruses in the absence of ad_juvants (saline control).

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One possible explanation for this result is that the initial 2 jig dose used for immunization was sufficient on its own for substantial responses to these large and complex antigens. In support of this notion is the widespread effectiveness observed in cats to this commercial FeLV vaccine (which is supplied without an adjuvant), and the lower overall responses seen with the 70 kDa heartworm protein, including the saline control. Not only is this antigen much smaller and less immunologically diverse than the virus preparations, only 1 /rg was used for immunization. For reasons that remain unclear, the primary responses to this immunogen were augmented most strongly by IFA. It is worth noting that since antigens coat the EIA test wells with varying efficiency and orientation, it is not possible to compare accurately the relative immunogenicity of different antigens by this direct binding method. This concern is not applicable in this study since the responses compared were from mice all of which received a particular antigen combined with different adjuvants. The lack of toxicity observed with GBA in mice, rabbits and cats is consistent with the broad dose response range observed in cell culture studies in which doses 10000 times that required to induce an effect were non-inhibitoryJ. The issue of safety is paramount given the regular occurrence of unacceptable sideeffects observed in otherwise potent immunomodulators. As expected, no adverse side-effects wcrc observed with either form of aluminum hydroxide when given alone. Further, the combination of GBA with HPA was well tolerated. The enhanced adjuvant effects of this combination of GBA with an aluminum hydroxide vehicle of proven safety would appear to be worthy of further study. The efficacy of this combination may best be demonstrated by exploiting the increased adsorptive capacity of RehydragelTM HPA with larger quantities of weaker immunogens.

ACKNOWLEDGEMENTS The author gratefully acknowledges the support of NIH SBIR grant #1 R43 AI29772-OlA1 and the funding and materials received from Reheis. The author also thanks Dr York of BioTrends International for the gift of the FeLV vaccine.

REFERENCES Glenny, A. T., Pope, C. G., Waddington, H. and Wallace, U. The antigenic value of toxoid precipitated by potassium alum. Journal of Pathology and Bacteriology 1926, 29, 38-45. Gupta, R. K., Rost, 6. E., Relyveld, E. and Siber, G. ft. Adjuvant properties of aluminum and calcium compounds. In: Vaccine Design: The Subunit and Adjuvant Approach (Eds Powell, M. F. and Newman, M.J.). Plenum Press, New York, 1995, pp. 229-242 Vogel, F. R. and Powell, M. F. A compendium of vaccine adjuvants and excipients. In: Vaccine Design: The Subunit and Adjuvant Approach (Eds Powell, M. F. and Newman, M. J.). Plenum Press, New York, 1995, pp. 141-227 Usinger, W. R. and Mishell, R. I. Chemical characterization and immunoenhancing activities of purified Cflophaga exopolymer. In: Vaccine Adjuvants and Modulators of Non-specific Resistance (Ed Majde, J. A.). Alan R. Liss, New York, 1987, pp. 125-137 Zeidner, N. S., Relasco, D. L., Dreitz, M. J., Frank, G. Ft. and Usinger, W. R. Gliding bacterial adjuvant stimulates feline

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cytokines in vitro and antigen-specific IgG in viva. Vaccine 1995,13,1294-1301. Shiigi, S. M., Capwell, Ft. Ft., Grabstein, K. H. and Mishell, R. I. Sera and the induction of immune responses. Ill. Adjuvant obtained from gliding bacteria with properties distinct from enteric bacterial lipopolysaccharide. Journal of Immunology 1977, 119,679-688. Allison, A. C. and Byars, N. E. Syntex adjuvant formulation. Research on Immunology 1992, 143, 519-525.

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Kensil, C. R., Barrett, C., Kushner, N., Neltz, G., Storey, J., Patel, U., Recchia, J., Aubert, A. and Marciani, D. Development of a genetically engineered vaccine against feline leukemia virus infection. Journal of the American Veterinay Medical Association 1991, 199, 1423- 1427. Alving, C. R., Detrick, B., Richards, R. L., Lewis, M. G., Shafferman, A. and Eddy, G. A. Novel adjuvant strategies for experimental malaria and AIDS vaccines. Anna/s of the New York Academy of Sciences 1993,690,2655275.

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