Biological features of genetic immunization

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Vaccine, Vol. 15, No. 8, pp. 788-791, 1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-41OW97 517+0.00

Biological features of genetic immunization Michael A. Barry and Stephen Albert Johnston* Genetic immunization (a.k.a. DNA-based immunization) shows promise at least as a convenient method to test and discover new vaccines and may be an eficient vaccine delivery system. However, relatively little is known about the parameters aflecting its eflectiveness, let alone its basic underlying biological mechanisms. Here we report on investigations of some of the factors that determine the quantity and quality of the immune response with genetic immunization. We find that for non-toxic proteins the antibody response correlates well with the level of expression as does the cellular response to a certain level. The augmentation of the immune response by co-introduction of a cytokine gene as a genetic adjuvant is also responsive to the expression level of the antigen. The immune response is inversely correlated to the age of the mice and at least part of this e#ect is through level of expression of the antigen. Gene gun administration of the transgene to the skin has the advantage over muscle injection in that ca loo-fold less DNA is required for the same level of expression and the injections are more reproducible in efect. Finally, the apparent dtflerences in Th2 (gun) vs Thl (muscle) responses between the two modes can at least partly be accounted for by dtflerences in the amount of plasmid DNA typically administered. 0 1997 Elsevier Science Ltd Keywords: genetic immunization;

DNA immunization;

vaccines;

gene gun

Genetic immunization looks very promising as a new way to administer vaccines. Yet, little is known about the biology underlying this technology. We have examined several variables affecting genetic immunization: (1) the antigen-its intracellular biology, expression level and toxicity; (2) the host animal-its genotype and age; (3) the method of gene inoculation-by gene gun or intramuscular (i.m.) injection; and (4) the amount of DNA used for inoculation. From this work, it is becoming clear that each element can effect the efficiency of genetic immunization, with the character of the antigen and host genotype being most dominant. As secondary issues, the amount of DNA used for inoculation can have as much influence on the strength and character of immune responses as the route of inoculation. Genetic immunization was first demonstrated by our laboratory in 1992l where simple plasmids encoding human growth hormone (GH) and human u-l antitrypsin (AAT) were inoculated into mice to produce humoral immune responses against these antigens. In this initial publication, we predicted that genetic immunization might provide a novel approach to vaccine development. This prediction has indeed been proven accurate. With each subsequent year, increasing numDepartments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, TX 752358573, USA. *To whom correspondence should be addressed.

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bers of papers are demonstrating the wide application of this technology against viral, bacterial, and parasitic pathogens and against cancer. In many cases, the cellular and humoral immune responses observed after genetic immunization have been long-lived, to the extent that antibodies and cytotoxic T lymphocyte (CTL) activity can be observed in mice years after single or multiple immunizations. While genetic immunization has proven remarkably effective as an approach to vaccination, little is known about the biology underlying this effectiveness. The following represents some of our laboratory’s observations and thoughts about several fundamental events and steps in performing genetic immunization effectively.

RESULTS AND DISCUSSION The role of expression level in genetic immunization There is some debate about the role of levels of gene expression in genetic immunization. The answer to this question is highly dependent on each antigen and whether cellular or humoral immune responses are the goal. It requires less antigen to elicit cellular responses than humoral responses in genetic immunization (gene gun humoral responses require -40 ng, for cellular ~1 ng). For secreted antigens such as AAT, we find a direct correlation between expression level and antibody production. When more CMV-AAT plasmid (cytomegalovirus promoter driving AAT expression) is inoculated, increasing levels of antibodies are produced (data

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Figure 1 Influence of expression level on genetic immunization. (A) Effect of insertion of an intron 5’ to the luciferase cDNA on luciferase expression in mice. Mice were inoculated with 2.5 pg twice using the gene gun in each ear with the indicated construct and luciferase activity was measured 18 h later. (B) Effect of insertion of an intron in antigen and genetic adjuvant plasmids. Mice were inoculated twice with 2.5 ,ug of each construct and AAT antibodies were measured 30 days later. (C) Effect of mouse age at inoculation on luciferase expression and AAT antibody production. Mice of the indicated age were inoculated twice with 2.5 ,ug CMV-LUC or CMV-AAT and luciferase activity was measured 18 h later or AAT antibody levels were measured 60 days later. (0) Expression level and variation following gene gun or i.m. injection. Mice were inoculated twice with 1 pg CMV-LUC in each ear and i.m. injected with 5Opg CMV-LUC in 50~1 PBS-MS into each tibialis anterior muscle. Luciferase activity was measured 18 h later and normalized to the amount of plasmid used

not shown). When an intron is inserted 5’ to luciferase in the same CMV plasmid backbone, expression increases fivefold (Figure IA). Similarly, insertion of an intron into CMV-AAT increases AAT antibody production two to threefold (Figure 1B). Increased expression has similar effects on the ability of granulocyte-macrophage colony stimulating factor (GM-CSF) to augment AAT antibody production (Figure 1B). For AAT, the titration of response with plasmid amount and expression strength makes sense, since the gene product is not particularly toxic and is continuously eliminated by secretion. Therefore more plasmid produces more antigen to be recognized by the immune system. This is in marked contrast to proteins like the rabies glycoprotein that when expressed at higher levels produce no increase in immune responses2. For this toxic protein and others like them, it is likely that increasing their expression merely kills the cells more quickly thereby blocking continued antigen production by the transfected cells. Many proteins from intracellular pathogens that are used in genetic immunization also modify or shut down host cell function and may cause similar negative effects in vaccines. For these antigens, there is no benefit in using plasmid vectors designed for maximum expression (i.e. CMV promoter with 5’ intron). For these types of problematic antigens, it may be advisable to break down these genes into sub-

domains to inactivate their toxicity prior to insertion into over-expression plasmids. There is some evidence that the level of expression is more crucial for cellular responses. For some antigens bearing CTL epitopes (e.g. HIV gp160), CTL responses titrate directly with decreasing amounts of DNA below 40 ng by gene gun. However, higher amounts of plasmid give lower CTL responses after single immunization (data not shown). This suggests that too vigorous a cellular immune response generated with large amounts of plasmid might attenuate vaccine responses, perhaps by killing off the transfected cells too early to maintain immune stimulation. The effects of mouse age on gene expression and antibody production

Although most investigators use 6 week or older mice for genetic immunization, little is known about what age of mice is optimal. When the luciferase reporter gene was inoculated into the epidermis of mice of various ages with the gene gun, there was an inverse relationship between age and the amount of gene expression (Figure ZC). Relative to 4-week-old mice, gene expression was 30% in 6-week-old mice and 14% in lo-week-old mice. If gene expression directly correlates with the level of immune responses observed, then this suggested that

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the common use of older mice might reduce immune responses. To test this, 4-, 6-, and lo-week-old mice were inoculated at a single time with CMV-AAT. In all age groups, antibody levels increased over 60 days and became stable thereafter (data not shown). As with luciferase expression, antibody production was inversely proportional to age being highest in the 4-week-old mice. Relative to 4-week-old mice, AAT antibodies were 77% in 6-week-old mice and 66% in lo-week-old mice. This indicates that immune responses are related to antigen expression levels and that some, but not all age-dependent immunization is due to variation in plasmid expression with age. This suggests that better immune responses can be achieved by inoculating younger animals rather than 6- to g-week-old mice. Gene gun vs intramuscular injection The first step in genetic immunization is transfection of cells in situ. Initially, it appeared that only muscle cells could take up naked DNA from solution and be transfected (as assessed by reporter gene activity)3. To transfect other tissues efficiently, a gene gun had to be used4. Subsequently, Robinson’s group showed that DNA injection into other sites [intravenous, intratracheal, intrabursal, intraorbital, intradermal (i.d.), subcutaneous51 could produce detectable immune responses, although under conditions where the actual level of transfection was probably low and the identity of the transfected cells are unknown. In that initial work by Robinson’s group, DNA delivery by direct i.m. injection required N 100 of times as much DNA as the gene gun to elicit equivalent immune responses. This and other observations has led to controversy over the relative effectiveness of the gene gun vs i.m. injection. We have conducted experiments to characterize these two methods of genetic immunization. There is a fundamental difference in these two methods in the amount of DNA required to produce an equivalent level of raw gene expression as demonstrated using reporter genes (Figure ID). On average, i.m. (Figure ID) and i.d. (data not shown) injection requires delivery of ca 100 times the amount of luciferase plasmid to produce an equivalent amount of reporter gene expression as produced by the gene gun. In addition, there is a higher likelihood of “misses” or inoculations having lO-lOO-fold lower expression by i.m. injection than by the gene gun (Figure ID). This apparent difference is not due to poor luciferase recovery from the muscle relative to the skin, since similar data is seen with peroxisomal vs cytoplasmic luciferase and when the amount of detergent is increased in the lysis buffer. This apparent difference in efficiency is most likely related to the fact that i.m. and i.d. injection places the DNA extracellularly where the majority of DNA is rapidly degraded by nuclease$. By contrast, the gene gun delivers DNA inside the cells, cirumventing this initial reduction in functional plasmids. Since most investigators have not quantitated what fraction of their injections are “good”, it is possible that animals may miss immunization rounds when single or only a few injections are made. This might explain some observations where a subset of animals are fully protected while others are totally susceptible. Dividing the inoculum into four injection sites may decrease the chance that escapes occur.

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Quantitative differences in immunization by i.m. and gene gun When the gene gun and i.m. injection have been directly compared in producing immune responses in our laboratory, i.m. injection has generally been less efficient, but not to the same degree as indicated by luciferase expression. Equivalent protection was observed with Mycoplasma pulmonis expression libraries when 16 times as much DNA by i.m. injection relative to gene gun7. However, for the secreted proteins AAT and GH inoculation of low amounts (2Spg) of plasmids encoding either antigen provoked similar antibody levels by both i.m. injection and gene gun delivery (data not shown). The efficiency of responses to antigens is also highly dependent on the genotype of the animals. We have observed differential responses in BalblC and C57BL/6 mice to both AAT and GH in the absence or presence of cytokine genes (data not shown). This is consistent with the genotype-specific protection observed in the mouse malaria model of genetic immunization*. This suggests that the efficiency of either route of genetic immunization is likely determined by the antigen itself (e.g. level of expression and efficiency of post-translational processing in the tissue) and the ability of the animal’s immune system to recognize the available epitopes. With these caveats, both routes of immunization can be routinely used with pg amounts of plasmid, but the gene gun remains more efficient for those cases where only small amounts of plasmid DNA would need to be used. Qualitative differences in immunization by the gene gun and i.m. injection It has been suggested that qualitative differences in the character of immune responses are elicited by epidermal and i.m. injection. In particular, it has been suggested that epidermal gene delivery elicits T,, responses while i.m. injection elicits T,, responses. This is probably an oversimplification, since the gene gun is not “restricted” to only TH2 responses.’ Indeed we and others have observed that the gun can produce strong CTL responses using nanogram’ and sub-nanogram amounts of plasmid DNA (data not shown). When we have directly compared isotypes of antibodies produced by the gun or i.m. injection using AAT or GH plasmids, we found that both elicit predominantly IgG, antibodies when only a few micrograms of DNA is used for the inoculum. What is most interesting, is that we could shift the isotypes to IgG,,, which is indicative of T,, responses, by merely increasing the amount of plasmid that was delivered by i.m. to a standard amount of 50 pug (data not shown). Similarly, gene gun delivery of 5Opg also increased IgG,, levels against AAT (not shown). This suggests that much of the observed preference of i.m. injection to promote T,, responses may be due to the adjuvant effects of the large amounts of bacterial plasmid DNA” generally used in this route of genetic immunization. Advantages of i.m. injection Although the gun may be more efficient, i.m. injection has its own advantages. First, it is considerably easier and faster to perform i.m. injection than using a gene gun. In addition, the cost of purchasing a gene gun is of

Biological features of genetic immunization:

course considerably higher than purchasing syringes and needles. Whether the higher costs associated with having to purify more plasmid for i.m. injection would ever equal the costs of buying a gene gun over time remains to be determined. The gun is also limited by a lower maximum amount of plasmid that can be delivered in one inoculation, For our gun in normal use in mice, this limit is 2.5,~g of plasmid per shot. Exceeding this amount causes the particles to clump together, creating “macroparticles” which cause increased damage to the target tissue. One can increase the amount of particles used to allow more DNA to be used, but damage can be caused by increasing particle density. By contrast, milligram quantities can be delivered by i.m. injections. In our hands, using large amounts of plasmid (2 50 pug) in single inoculations produces higher antibody levels to AAT or GH than can be achieved by single inoculation with the gun. In conclusion, our initial results indicate that a hierarchy of elements exist in producing good immune responses by genetic immunization. Predominant among these factors is the biology and epitopes of the antigen and the genotype and age of the host. The gene gun appears to be more efficient for eliciting immune responses per unit DNA, but does entail a substantial economic and learning investment. By contrast i.m. injection is simple and may be able to elicit higher immune responses by virtue of being able to deliver very large amounts of plasmid to the animal. Qualitative issues between the gun and i.m. injection still need sorting out, but preliminary data from our laboratory suggests that at least some of the reported differences may be due to the amount of DNA used in each method.

M.A. Barry and S.A. Johnston

REFERENCES 1

Tang, D., DeVit, M. and Johnston, S.A. Genetic immunization is a simple method for eliciting an immune response. Nature 1992, 356, 152-l 54 2 Xiang, Z.Q., Spitalnik, S.L., Cheng, J., Erikson, J., Wojczyk, 6. and Em, H.C.J. Immune responses to nucleic acid vaccines to rabies virus. Virology 1995, 209, 569-579 3 Wolff, J.A., Malone, R.W. and Williams, P. et a/. Direct gene transfer into mouse muscle in vivo. Science 1990, 247, 1465-1468 4 Johnston, S.A., Riedy, M., DeVit, M.J., Sanford, J.C., McElligott, S. and Williams, R.S. Biolistic transformation of animal tissue. In Vifro Cell. Dev. Viol. 1991, 27P, 11-14 5 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. Proc. Nat/ Acad. Sci. USA 1993, 90, 11478-11482 6 Levy, M.Y., Barron, L.G., Meyer, K.B. and Szoka, F.C. Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into the blood. Gene Ther. 1996, 3, 201-211 7 Barry, M.A., Lai, W.C. and Johnston, S.A. Protection against mycoplasma infection using expression library immunization: a general approach to vaccine development. Nature 1995, 377, 632-635 M., Hedstrom, R.C., Hobart, P., 8 Doolan, D.L., Sedegah, Charoenvit, Y. and Hoffman, S.L. Circumventing genetic restriction of protection against malaria with multigene DNA immunization: CDB+ T cell-, interferon T-, and nitric oxide-dependent immunity. J. Exp. Med. 1996, 163, 1739-1746 9 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 10 Krieg, A.M., Yi, A.K. and Matson, S. et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995, 374, 546-549

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