Manipulation of HIV-1 gp120-specific immune responses elicited via gene gun-based DNA immunization

PII: SO264-410X(97)00036-4

Vaccine, Vol. 15, No. 12113, pp. 1349-1352, 1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X197 $17+0.00

ELSEVIER

Manipulation of HIV-l gpl20-specific immune responses elicited via gene gun-based DNA immunization Sudhirdas

K. Prayaga*$,

Maura

J. Ford*

and Joel R. Haynes*“f

Gene gun-based DNA immunization using vectors encoding HIV1 gp120 or influenza virus nucleoprotein result in Th2-like immune responses following successive immunizations. The codelivery of vectors encoding IL-2, IL- 7, or IL-12 blocked this effect by markedly enhancing gpl20-specific inte$eron gamma production, and suppressing IL-4 and IgGl responses. An unbiased augmentation of all immune responses was observed by increasing the resting period between immunizations. In this case, IFN-~1 production following in vitro stimulation increased by over lOOO-fold, while IL-4, IgGl, and IgG2a responses were elevated as well. Interestingly, cytokine gene codelivey, in the context of the longer resting period, provided no additional stimulation of Thl-like responses such as IFN-7 and IgG2a production, although there was still some suppression of IL-4 production. These data demonstrate that the quality and magnitude of responses elicited following epidermal administration of DNA vaccines can be manipulated by multiple means. 0 1997 Elsevier Science Ltd. Keywords:

DNA tion

DNA

immunization,

immunization

gene

represents

gun,

an

HIV-l

gp120

attractive

vaccina-

strategy

for the induction of humoral, cellular, and protective immune responses specific for a variety of infectious and parasitic disease antigens’-lY. The ability to mimic the actions of live vaccines by the induction of cytotoxic cellular responses, as well as antibody responses specific for conformational determinants, and the simplicity and commonality of this approach for diverse pathogens, are compelling reasons to move this technology into human clinical evaluation. Recent activity in the characterization and manipulation of immune responses following direct DNA delivery in viva in mouse models has demonstrated that direct intradermal and intramuscular inoculation of plasmid DNA results in the induction of T helper l-like (Thl) responses characterized by IFN-?; production and predominantly IgG2a antibodies’x-‘3. More recently, this phenomenon was shown to be dependent upon adjuvant properties of the specific CpG-containing bacterial DNA sequences, termed immunostimulatory sequences (ISS), that result in enhanced immunogenicity, with referential augmenta,P tion of Thl cytokine production--. In contrast to intradermal and intramuscular inoculation, direct intracellular DNA delivery to epidermal *Geniva,

8585 Science Dr. Suite C Madison, WI 53711, USA. tTo whom correspondence may be addressed. Present address: Heska Corp., 1825 Sharp Point Drive, Fort Collins, CO 80525, USA. *Present address: HoffmanLa Roche, Dept. Immunology, Nutley, NJ 07110, USA. (Received 22 October 1996; revised version received 13 January 1997; accepted 14 January 1997)

keratinocytes using a gene gun elicits antibody responses in mice that consist mainly of the IgGl subclass, as well as cytokine responses that shift to progressively stronger T helper 2-like (Th2) profiles (IL-4 > IFN-7) with successive immunizations”. ‘. This phenomenon may be related to the fact that immune responses elicited via epidermal gene gun delivery are not significantly affected by the adjuvant properties of bacterial plasmid DNA that are apparent following parenteral inoculation. This idea is also supported by the observations that strong responses can be elicited by gene gun delivery using very small quantities of DNA (9) the IgGl-to-IgG2a ratio is not affected by titration of the DNA inoculum from 2.5 pg to 40 /lg (T. R. Roberts and J. R. Haynes, unpublished), and immune responses in rodents and pigs are not diminished when plasmid backbones lacking ISS elements are substituted (manuscript in preparation). Inasmuch as the route of DNA delivery (gene gun versus parenteral inoculation) influences the quality and types of immune responses elicited, the potential to further mani ulate these responses via cytokine gene codelivery’ B,w* or the inclusion of adjuvants*’ continues to be an intriguing possibility. Previously, it was shown that the codelivery of vectors encoding a cytokine such as GM-CSF can augment antigenspecific responses following both intramuscular or epidermal plasmid DNA deliveryz0,29. However, the potential to qualitatively modulate the types of responses elicited (i.e. Thl vs. Th2) by cytokine vector codelivery has not been described. In this report we demonstrate that vectors encoding interleukins -2, -7, and -12 can independently enhance

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antigen-specific IFN-7 production and suppress both IgGl and IL-4 responses, when codelivcrcd with an HIV-I gp120 vector to the epidermis via gcnc gun treatment. Interestingly, an unbiased augmentation of immune responses was observed when, in the absence of cytokinc gene codelivcry, the resting period between gene gun immunizations was Icngthcned. These data are consistent with the potential to manipulate the types of responses elicited via gent gun-mediated DNA immunization, and suggest that the particular dosing regimen can have marked effects.

MATERIALS

AND

METHODS

Immunizations n-g-week-old female BalbiC mice (Harlan-SpragueDawley, Indianapolis, IN) received gene gun-mediated DNA immunizations employing a gp120 expression vector”, as described by Pertmer et al.” using the hand-held, helium powered, Accell” gene delivery system (Geniva, Middleton, WI). The murine IL-12 vector was as previously described”“. The vector encoding murine IL-2 was derived from a cDNA clone (pmutl) obtained from American Type Culture Collection (Rockville, MD), and inserted into a human cytomegalovirus (hCMV) immediate early promoterbased expression vector essentially identical to one previously described”. The IL-7 vector was derived by PCR cloning of a murine IL-7 coding sequence from murinc splcnocyte cDNA, which was inserted into the hCMV expression vector.

Measurement of antigen specific IL-4 and IFN-;I production Measurement of Gpl20-specific IFN-7 and IL-4 production in l&o by gp120 peptide-stimulated splenocytes was performed as previously described’“. In the case where purified T cells rather than total splenocytes were employed, T cells were purified via passage wool through columns (Poly Sciences, nylon Warrington, PA). In such T cell cultures, naive syngeneic spleen cells coated with the 15mer gp120 peptide (RIQRGPGRAFVTIGK), and fixed with mitomycin-C, were used as stimulators. Conditioned media containing the secreted cytokines were collected form all cultures after 48 h. The levels of IL-4 and IFN-7 in the conditioned media were measured as previously described’“.

Measurement of serum anti-gp120 IgGl and IgG2a levels gpl20-specific IgGl and IgG2a responses were measured as previously described” except that recombinant HIV-l gp120 (Intracel, Cambridge, MA) was used to coat the EIA plates. Actual levels of gpl20-specific IgGl and IgG2a were quantified by use of purified IgGl and IgG2a standard curve samples that were either coated directly onto ELISA plates or were captured with a pre-coated goat anti-mouse Ig capture antibody (Southern Biotechnology Associates). The IgGl and IgG2a assays exhibited essentially identical sensitivities.

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SK. Prayaga et al. RESULTS

AND

DISCUSSION

To determine if cytokine gene codclivcry could modulate the quality of responses elicited via gcnc gun-mediated DNA immunization, a scrics of mice received either 1. 2, or 3 consecutive cpidermal DNA immunizations consisting of an HIV-1 gp120 cxprcssion vector. with or without an additional vector encoding murine IL-,,7 -7, or -12. Successive immunizations were at 1 month intervals and animals in each group wcrc sacrificed 2 weeks following their final immunization. Gp120-specific IFN-1’ and IL-4 production lcvcls were measured in an in vitro antigen stimulation assay in the absence of exogenous cytokinc addition. Serum samples wcrc also collected for quantilication of the lcvcls of gpl20-specific IgGl and lgG2a antibodies. 7?ih/c 1 shows the IgGl and lgG2a responses for those animals that received tither two or three gp120 DNA immunizations (boost 1 and boost 2. respectively). In the absence of cytokine vector codelivery, gp l20-specific IgG 1 responses increased by l&fold between the first and second booster immunizations, while IgG2a responses incrcascd by a lesser extent (n-fold). These data arc consistent with our earlier results demonstrating an apparent Th I -toTh2-like shift in gp120 and influenza nucleoproteinspecific responses with successive immunizations’g.“. this bias towards IgGl responses Interestingly, following the third immunization was significantly suppressed by the codelivery of each of the cytokinc ( 1L-2, P = 0.037; IL-7. P = 0.016; IL-12, vectors P = 0.04Y). However. lgG2a responses were not rcciprocally augmented by cytokine gene codclivcry. IgG1 and IgG2a responses were also measured in 4 separate groups of mice that received primary immunizations only, in the prcscnce or absence of cytokinc vector codelivery. Consistent with our earlier results”, significant gpl20-specific humoral responses were not detected at either 4 or 12weeks following a primary immunization (data not shown). More compelling evidence for modulation of the quality of gp120-specific responses via cytokinc gene codelivery was obtained from the IFN-y and IL-4 production patterns (Tahlr 2). Using the gp120 vector alone, mean gp120-specific IFN-7 production levels were weak in animals that received a primary immunization only, and below detection following the booster Table 1 Measurement of HIV-l gpl20 specific IgGl and IgG2a antibody levels in DNA-immunized mice with and without cytokine vector codelivery. All mice received epidermal gene gun immunizations consisting of 1 ,rg of an HIV-l gp120 expression vector either alone, or in combination with 1 /(g of an IL-2, IL-7 or IL-12 vector as indicated. Four groups of four mice each were immunized twice at 0 and 4 weeks (Boost 1 data), while an additional four groups of four mice each received three immunizations at weeks 0, 4 and 8 (Boost 2 data). HIV-l gp120 specific IgGl and IgGZa levels were measured 2 weeks following the last immunization

Cytokine used

IgGl” ~ Boost 1

None IL-2 IL-7 IL-12

3720 f 2523 8623 i 2583 4924+1894 1833+1071

Boost 2

lgG2” ~ Boost 1

Boost 2

68 142 f 2955 27 872 f 14,964 30373*10,569 36182*13,105

687+324 998 i 518 820+197 937f226

4082+892 4238 & 1611 3232+1072 4337k623

a Antibody levels expressed as ng ml of serum f SEM.

Manipulation

of gene gun-elicited

Table 2 Measurement of antigen specific IFN-;I and IL-4 production in DNA-immunized mice following cytokine DNA codelivery. Immunizations were performed as described in Table 1, but included an additional set of mice immunized only once. Spleen cells collected at weeks 4, 6, and 10 following one, two or three immunizations, respectively, were used to measure IFN-; and IL-4 production following in vitro stimulation with recombinant HIV-1 gp120 interferon

;,(ng/ml)”

IL-4 (pg/ml)b

Cytokine used

Primary

Boost 1

Boost 2

Boost 1

None IL-2 IL-7 IL-12

11.7k4.7 28.9 f 4.5 21.6f12.5 10.5k7.6

0 191 k51.8 75 + 7.4 210+53.5

0 292+163 48k21.3 102i80

32i3.4 16i4.2 0 0

a measured b measured

in supernatants

of total splenocytes.

in supernatantsof nylon wool-purifiedT ceils.

immunizations. Again, these results are consistent with our earlier work demonstrating diminishing IFN-1, immunizati0ns’4.2J. production with successive

cytokine vector codelivery resulted in However, markedly enhanced IFN-7 production in vitro, in animals that received one or two booster immunizations. Consistent with these results, IL-4 production in vitro was suppressed in supernatants derived from animals that received cytokine vector codeliveries. In the latter case, IL-4 activity was not detected in direct splenocyte supernatants, but was detected in the supernatants of nylon wool-puritied T cells from boost 1 animals following antigen stimulation (T cells were not purified from the splenocytes of animals from the primary and boost 2 immunization groups). Interestingly, IL-4 activity was only detected from purified T cells of animals that received the gp120 vector alone or the gp120+ IL-2 vectors. IL-7 and IL-12 vector codelivery resulted in a complete suppression of detectable IL-4 production from purified T cells. These data, when combined with the IFN-7 and IgG isotype data, demonstrate that cytokine vector codelivery can block the tendency of gene gun-based DNA vaccines to elicit Th2-like responses. Since our earlier reports are consistent with the possibility that multiple and frequent immunizations are at least partially responsible for the Th2 bias demonstrated for gene gun immunization”‘.“, the possibility of modulating these responses by administering fewer immunizations over a longer time period was investigated. To this end, additional groups of mice in the experiments described above received only two epidermal DNA immunizations consisting of an HIV-l gp120 expression vector, with or without an additional vector encoding murine IL-2, -7, or -12. However, in this case, the resting period between the two immunizations was extended to 3 months. Table 3 shows that in each immunization group, humoral responses were measurably increased when animals were boosted after a 3 month rest versus a 1 month rest (compare with Table I, Boost 1 data), however, these increases were of borderline significance except for the increase in IgG2a responses in the absence of cytokine vector codelivery (P = 0.02). In no case did cytokine vector codelivery result in any decrease in the IgGl:IgG2a ratio as was observed following the second boost in the short rest period groups (Table I).

immune responses:

S.K. Prayaga et al.

Table 3

Measurement of HIV-1 gp120 specific antibody and cytokine responses after one booster immunization in long rest period animals. Four groups of four mice each received two epidermal gene gun immunizations at weeks 0 and week 12 containing 1 119 of the gp120 expression vector either alone, or in combination with 1 j/g of an IL-2, IL-7 or IL-12 vector as indicated. HIV-l gp120 specific IgGl and lgG2a levels were quantified 2 weeks following the second immunization. IFN-;, and IL-4 production by antigen-stimulated total splenocytes was also measured 2 weeks following the second immunization Cytokine used

IgGl”

IgG2aa

IFN-;, (ng ml)

IL-4 (pg ml)

None IL-2 IL-7 IL-12

8954k3150 14,098i 1209 10,928 f 2824 5146+2555

185Ok269 1627i561 1375 f 320 807+448

13,299+1007 5122k2588 2113+501 11,799*1338

82k9.6 41+12.4 53 f 20.4 46k8.5

“antibody

levels expressed

as ng/ml serum f SEM.

In contrast to the modest changes in antibody responses, more compelling evidence for the potential of longer resting periods to influence gplZO-specific immune responses can be seen from the IFN-7 and IL-4 data (Table 3). By increasing the resting period to 3 months, IFN-~7 production following the booster immunization increased by 100 to lOOO-fold relative to any of the responses observed in animals that received the more aggressive immunization regimen (Table 2). In addition, IL-4 production increased as well. In the latter case, IL-4 activity was detected in direct supernatants from antigen-stimulated splenocytes at levels higher than those observed in the short rest-period experiment using purified T cells. Thus, by simply increasing the resting period from 1 to 3 months, the magnitude of the gpl20-specific cytokine responses following two doses was dramatically enhanced. These data, along with previous results’“.“, are consistent with the idea that the number of doses, rather than time, influences the quality of the responses following epidermal gene gun immunization, while elongation of the resting period between doses may increase the magnitude of a given response. It is interesting to note, that in the context of the longer resting period, cytokine gene codelivery provided no additional stimulus in Th 1-associated Rather, IFN-7 responses were markedly responses. reduced by IL-2 and IL-7 vector codelivery, and were not enhanced by IL-12 vector codelivery, suggesting that cytokine manipulation of immune response quality may be more difficult under conditions where antigenspecific responses have been augmented in general. Thus, the future role that cytokine gene codelivery may play in DNA immunization strategies will require further investigation. The dramatic enhancement in IFN-7 production in the context of the longer period is not inconsistent with our earlier reports, demonstrating a Th2 bias in elicited gene antigen-specific responses via gun-mediated DNA immunization, since IL-4 production was markedly enhanced as well. Because of the dominance of IL-4 over Thl cytokine8, it can still be argued that, even in the context of longer resting periods, gene gun-mediated DNA immunization leads to responses with significant Th2 character due to IL-4 and IgGl production. Indeed, the only reports of significant antigen-specific IL-4 production following DNA immunization have been associated with the gene gun

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route’y,2J, since direct intramuscular or intradermal inoculation of naked DNA results specifically in Thl responsesiX-“. This observation indicates that gene gun-based DNA immunization via the epidermis may be uniquely suited for the elicitation of antigen-specific anti-inflammatory immune responses, as compared to other routes of direct in ~ivo DNA delivery. Immune responses of this nature might bc more desirable for eliciting protection against mucosally transmitted viral diseases and certain inflammatory autoimmune disorders such as rheumatoid arthritis.

REFERENCES 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Tang, D.C., Devit, M.J. and Johnston, S.A. Genetic immunization is a simple method for eliciting an immune response. Nature 1992, 356, 152-154. Ulmer, J.B., Donnelly, J.J. and Parker, SE. et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 1993, 259, 174551749. Michel, M.L., Davis, H.L., Schleef, M., Mancini, M., Tiollais, P. and Whalen, R.G. DNA-mediated immunization to the hepatitis B surface antigen in mice: aspects of the humoral responses mimic hepatitis B viral infection in humans. Proceedings of the National Academy of Sciences, USA 1994, 92,5307-5311. Eisenbraun, M.D., Fuller, D.H. and Haynes, JR. Examination of parameters affecting the elicitation of humoral immune response by particle bombardment-mediated genetic immunization, DNA Cell Biology 1993, 12, 791-797. Robinson, H.L.. Hunt, L.A. and Webster, R.G. Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine 1993, 11, 957-960. Cox, G.J.M.. Zamb, T.J. and Babiuk, L.A. Bovine herpesvirus 1: immune responses in mice and cattle injected with plasmid DNA. Journal of Virology 1993, 67, 5664-5667. Fynan, E.F., Webster, R.G., Fuller, D.H., Haynes, J.R., Santoro, J.C. and Robinson, H.L. DNA vaccines: protective immunizations by parenteral, mucosal and gene gun inoculations. Proceedings of the National Academy of Sciences, USA 1993, 90, 11478-11482. Xiang, Z.Q., Spitalnik, S., Tran, M., Wunner, W.H., Cheng, J. and Ertl, H.C. Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus. Virology 1994, 199, 132-l 40. Pertmer, T.M., Eisenbraun, M.D., McCabe, D., Prayaga, S.K., Fuller, D.H. and Haynes, J.R. Gene gun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responses following epidermal delivery of nanogram quantities of DNA. Vaccine 1995, 13, 1427-1430. Sedagah, M., Hedstrom, R., Hobart, P. and Hoffman, S.L. Protection against malaria by immunization with plasmid DNA encoding circumsporozoite protein. Proceedings of the National Academy of Sciences, USA 1994,91,9866-9870. Raz, E., Carson, D.A. and Parker, S.E. et a/. lntradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses. Proceedings of the National Academy of Sciences, USA 1994, 91,9519-9523. Wang, B., Ugen, K.E. and Srikantan, V. et al. Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proceedings of the National Academy of Sciences, USA 1993,90,4156-4160. Yankauckas. M.A., Morrow, J.E. and Parker, S.E. et al. Longterm anti-nucleoprotein cellular and humoral immunitv is induced by intramuscular injection of plasmid DNA containing NP acne. DNA Cell Biology 1993. 12, 771-776. Wan-g, B., Boyer, J. and%ikantan, V. et al. DNA inoculation induces neutralizing immune responses against human immunodeficiency virus type 1 in mice and nonhuman primates. DNA Cell Biology 1993, 12, 799-805. Montgomery, D.L.. Shiver, J.W. and Leander, K.R. et a/. Heterologous and homologous protection against influenza A

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S.K. Prayaga et al

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

by DNA vaccination: optimization of DNA vectors, DNA Cell Biology 1993, 12, 777-783. Irwin, M.J., Laube, L.S. and Lee, V. et al. Direct injection of a recombinant retroviral vector induces human immunodeficiency virus-specific immune responses in mice and nonhuman primates. Journal of Virology 1994, 68, 5036-5044. Prayaga. SK., Fuller, D.H., Corb, M.-M. and Haynes, J.R.. Particle-mediated nucleic acid immunization. In Vaccine 95: Molecular Approaches to the Control of tnfecfious Diseases eds. R.M. Chanock, F. Brown, H.S. Ginsberg and E. Norrby. Cold Spring Harbor Laboratory Press, Plainview, NY, 1995, pp. 105-109. Manickan, E., Rouse, R.J., Yu, Z., Wire, W.S. and Rouse, B.T. Genetic immunization against herpes simplex virus: protection is mediated by CD4+ T lymphocytes. Journal of fmmunology 1995, 155, 259-265. Pertmer. T.M., Roberts, T.R. and Haynes, J.R. Influenza virus nucleoprotein-specific immunoglobulin G subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vector DNA delivery. Journal of Virology 1996, 70, 6119-6125. Xiang, Z.Q. and Ertl, H.C. Manipulation of the immune response to a plasmid-encoded viral antigen by coinoculation with plasmids expressing cytokines. fmmunity 1995, 2, 129-135. Raz, E., Tighe, H. and Sato, Y. et al. Preferential induction of a Thl immune response and inhibition of specific IgE antibody formation DNA immunization. by plasmid Proceedings of the National Academy of Sciences, USA 1996, 93,5141-5145. Hsu, C.H., Chua, K.Y. and Tao, M.H. et al. Immunoprophylaxis of allergen-induced immunoglobulin E synthesis and airway hyperresponsiveness in vivo by genetic immunization. Nature Medicine 1995, 2, 540-544. Sato, Y., Roman, M. and Tighe, H. et al. lmmunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 1996, 273, 352-354. Fuller, D.H. and Haynes, J.R. A qualitative progression in HIV type 1 glycoprotein 120. specific cytotoxic cellular and humoral immune responses in mice receiving a DNA- based glycoprotein 120 vaccine. AlDS Research in Human Retroviruses 1994, 10, 143331441. Kriesel, J.D., Spruance, S.L., Daynes, R.A. and Araneo, B.A. Nucleic acid vaccine encoding gD2 protects mice from herpes simplex virus type 2 disease. Journal of lnfecfious Diseases 1996, 173,536-541. Prayaga, SK. and Haynes, J.R. Cytokine regulation of HIV-1 gpl20specific immune responses via gene gun-mediated nucleic acid immunization. In Vaccine 96: Molecular Approaches to the Control of infectious Diseases eds. F. Brown, E. Norrby, D. Burton and J. Mekalanos. Cold Spring Harbor Laboratory Press, Plainview, NY, 1996, pp. 99-102 Raz, E., Watanabe, A. and Baird, S.M. et al. Systemic immunological effects of cytokine genes injected into skelet al muscle. Proceedings of the National Academy of Sciences, USA 1993,90,4523-4527. Leong, K.H., Ramsay, A.J., Boyle, D.B. and Ramshaw, I.A. Selective induction of immune responses by cytokines coexpressed in recombinant fowlpox virus. Journal of Virology 1994, 68,8125-8130. Conry, R.M., Widera, G. and LoBuglio, A.F. et al. Selected strategies to augment polynucleotide immunization. Gene Therapy 1996, 3, 67-74. Rakhmilevich, A.L., Turner, J. and Ford, M.J. et al. Gene gun-mediated skin transfection with interleukin-12 gene results in regression of established primary and metastatic murine tumors. Proceedings of the National Academy of Sciences, USA 1996,93,6291-6296. Chapman, B.S., Thayer, R.M., Vincent, K.A. and Haigwood, N.L. Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells. Nucleic Acids Research 1991, 19, 3979-3986. Swain, S.L., Weinberg, A.D., English, M. and Huston, G. IL-4 directs the development of Th2-like helper effecters. Journal of Immunology 1990,145,3796-3806.