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Vaccine strategies: selective elicitation of cellular or humoral

immunity? Gene M. Shearer and Mario Clerici

The discovery of immunoregulatory cytokines and the fact that they modulate the

cellular and humoral arms of the immune system, generally in opposing directions

raises fundamental questions concerning vaccine development. Because antibiotic-

resistant infectious organisms are appearing at an increasingly rapid rate, more

emphasis will need to be placed on prevention of infection/disease via immunization

and less on post-infection antibiotics. To accomplish this task effectively, immune

regulation should be integrated into vaccine design. Here we consider the opposing

potentials of cytokine-regulated cellular and humoral immunity, and question whether

the 'best of both worlds' is possible or desirable.

The immune system can be divided into two broad functional categories: humoral immunity (HI) and cel- lular immunity (CI) (1Kef. 1). HI is effected by anti- bodies, bivalent protein molecules that can be cell- bound or cell-free. Antibodies bind to antigenic determinants, usually on the surface of viruses, bac- teria or parasites, and inactivate these infectious agents. Antibodies are primarily effective against extracellular infectious agents. By contrast, CI is mediated by effec- tor cells that destroy infected cells of the host by direct cell-to-cell contact, or by the release of molecules that possess killing activity. In this way CI mechanisms focus on destruction of the source of infection, and are more effective against intracellular infections. CI is less clearly understood than HI and appears to be more complex.

Both CI and HI require a special type of T cell to initiate and/or enhance cellular and humoral responses. These cells are called T helper (Th) and pro- duce protein molecules known as cytokines that are responsible for the helper effects. Thus, both the HI and CI components of the immune system are depend- ent on Th, which actually comprises part of the cellular arm of immunity. Using cell cloning tech- niques, Mosmann and Coffman originally identified two functionally-distinct Th subsets in the mouse 2. These cloned helper cells, called Th l and Th2

G. M. Shearer (shearerg@dclOa.nci.nih.gov) is at the Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. M. Clerici (ma~o@imiucca,~i. unimi.it) is at the Cattedra di hnmunologia, Universita degli Studi di .~lilano, Milano, Italy.

cells produced, respectively, interferon-'y (IFN-y) and interleukin-4 (IL-4), and provided helper activity primarily for CI and HI. Similar findings were subse- quently made in the human 3. These above investi- gators and their colleagues also found that T h l and Th2 responses were regulated not only in a positive but also a negative way such that cellular responses were augmented by IL-2 and IFN- ' , /but decreased by IL-4 and IL-10. In contrast, antibody responses were enhanced by IL-4 and IL-10, but were down- regulated by IFN-~/4 and another cytokine, lL-12, that enhances IFN-y and is produced by monocytes s. The above summary, however, is oversimplified in that not all antibodies are enhanced by Th2 clones. For exam- pie, the IgG2a subclass can be enhanced by Th l clones.

Because these regulatory cytokines can be produced by different cell types and the in situ human immune system is more complex than a clone of T cells, we have interpreted and expanded the T h l / T h 2 hypoth- esis to the type 1/type 2 model that includes all cell types that produce these cytokines 1. Thus we characterize the immune response of an individual on the basis of whether there is a dominant cellular or humoral response with the associated production of IL-2, IL-12 and/or IFN-',/ or of IL-4, IL-5, IL-6 and/or IL-10, respectively (see Fig. 1). Box 1 also compares the T h l / T h 2 model that is based on cloned CD4 + T cells with the type 1/type 2 model derived from the concept that multiple cytokines produced by different cell types contribute to the overall balance or dominance of cellular or humoral immunity.

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IFNq, 11_-2

I FN-7

crossregulation

IL-4 IL-4 IL-5 IL-6 IL-10

Type 1 help

IFN-7 IL-2 IL-12

IFN-y IL-12

crossregulation -ql

IL-4 IL-10

' Type 2 help

IL-4 IL-5 IL-6 IL-10

Dominant Dominant Dominant Dominant cellular humoral cellular humoral

Figure 1 Corr~parison of the Thl/Th2 model of cloned CD4 ÷ T cells with the type 1/type 2 model for generation of dominant cellular or humoral responses.

Kinetics o f cellular and humora l responses The primary immune response against antigens of

int~,ctious agents characteristically involves a cellular effc ctor response followed by a humoral response (Fig. 2a) A typical secondary response resulting from previous immunization (either natural or induced) is shown in Fig. 2b. The secondary response develops more rapidly and is more potent than the primary response. The decline in CI that occurs concomitantly with the increase in HI may be owing to the down- regalatory potential of type 2 cvtokines, such as IL-4 and IL-10, that not only increases HI but also can reduce C1. This decline in CI during progression of the immune response may have t~vo important effects. Fir:.it, it may serve to keep the CI in check, thereby reducing the possibility of CI and type 1 cytokine- induced autoimmune conditions, for example Crohn's disease and type 1 diabetes. However, ifCI is the major protective arm of the immune system, such as in chronic intracellular infections, the shift from a domi- nant cellular to humoral response could compromise prcrtective CI, resulting in recurrence of an infection, to which the cellular arm of the innnune system had iniually gained control 6. Primary infection with HIV may be such an example, because the kinetics of cel- lular and humoral responses are similar to those of Fig. 2a and the serum viral titer drops by a factor of 102-104 just: after the acute infection phase, at the time of mare- mum cellular effector activity- and prior to the appear- ante of neutralizing antibody and the decline in CI (P,, :f. 7).

Figure 2c also shows a situation in which imnmn- izadon can result in CI without, or with minimal, HI. Selective immunization of the cellular arm has been seen in both animals ~,9 and humans, including immun- ization with HIV antigens m,11. This example of selec- tive immunization of CI is characterized by low dose antigen exposure or infection, and could provide an approach for preferential immunization of C1 (R, efs 12 13). We are not aware of dose-related immuniz- ati, m conditions that elicit HI without also inducing CI. although the kinetic patterns shown in Fig. 2a and 2b suggest that antibodies can dominate later in the immune response.

Condit ions for cellular versus humora l i m m u n i t y Until very recently, vaccine strategies have not con-

sidered the design of vaccines that would preferentially elicit either CI or HI. Therefore, published infor- mation on this topic is limited. Box 2 lists several pa- rameters that may contribute to a dominant cellular or humoral response, although the conditions under which some of these parameters result in a preferen- tial response of one Wpe or the other have not yet been determined.

As mentioned above, low dose immunization can result in a dominant cellular response without appre- ciable antibody production. This phenomenon has been observed in non-viral animal models s and in macaques exposed to SIY (Ref. 9). Immunization with higher doses of antigen results in both CI and HI, with HI often persisting after CI has waned (see Fig. 2). Notably, low dose inoculation of mice with proto- zoan parasites followed by high dose challenge has been reported to result in only a cellular response,

Box 1. Comparison of T h l / T h 2 and type 1/type 2 models

Model Cellular source of cytokines

• Thl/Th2 • CD4 ÷ T

• Type 1/ • CD4 +T;CD8 +T; type 2 monocytes; B cells

Description of model

• Tests for cytokines produced by cloned CD4 ÷ T cells • Tests cytokine effects produced by multiple cell types on immune function

Box 2. Factors in vaccine design that can influence dominant cellular versus humoral responses

• Dose of antigen • Adjuvant used • Route of immunization • Type of antigen • Type of antigen presenting

cells

• Costimuiatory signals • Genetic background of vaccinee • Cytokine environment • Immunologic status of vaccinee • Age of vaccinee

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g

} O9

.{

a

CI HI

c

Cl

f HI

j l

Relative time

Figure 2 Kinetics of cellular immunity (CI) and humoral immunity (HI). (a) Char- acteristic primary immune response illustrating dominant CI followed by dominant HI. (b) Typical secondary immune response showing similar dominant CI followed by dominant HI, but with more rapid kinetics and stronger responses than in primary response. (c) Atypi- cal strong and persistent CI in the absence of or with weak HI, result- ing from low-dose antigen immunization.

despite the fact that a primary high dose response elic- its a dominant HI (1Kefs 14, 15). This phenomenon has been termed 'low dose lock-in', and suggests that, once initiated, a selective cellular response is not eas- ily switched to a strong humoral response. Thus, a mouse strain that is genetically susceptible to leish- maniasis and to tuberculosis can be protected by low dose immunization and CI against subsequent high infectious doses that would normally result in antibody production and disease 1<15. It is possible that HIV- exposed individuals who remain seronegative but exhibit cellular responses were initially exposed to a low dose of HIV antigens, and that HIV-specific CI was dominant, despite subsequent exposures to high viral doses that might otherwise elicit a strong humoral response13.

Adjuvants can contribute to directing a response towards dominant CI or HI. Alum, the major adju- vant approved for human immunization, is known to elicit strong antibody responses. Adjuvants based on bacterial products, such as mycobacteria, are consid-

ered to be strong potentiators of cellular responses. Other adjuvants are currently under study for their ability- to elicit dominant CI or HI or both types of immunity. Antigens presented in liposomes have been found to elicit strong CI, and monophosphoryl lipid A has been reported to be an adjuvant that generates strong and persistent cellular and humoral immunity 16. Cytokines can also be used as adjuvants administered either separate from the vaccine or inserted as part of the genetic element of the immunizing material.

The type of antigen-presenting cells (APCs), prepar- ation of the vaccine, route of immunization and co- stimulatory signals can all influence dominant cellular or humoral responses. It is generally considered that APCs of the monocyte/macrophage lineage present antigen that favors dominant CI, whereas B cells present antigen that elicits dominant HI (IKef. 17). Genetic background of the vaccinee may also influ- ence CI and HI. For example, the BALB/c and C57BL/6 inbred mouse strains exhibit dominant humoral and cellular responses after infection with sev- eral different parasites and bacteria 15. However, it is doubtful that such distinct genetic differences will be observed in outbred humans. Age may also influence immune potential, as well as CI and HI. It appears that the potential for dominant humoral immunity may exist in infants and possibly in aged animals and humans 18.

Several different types of vaccine preparations have been used for immunization, including whole killed organisms, synthetic peptides of antigens from infec- tious agents, attenuated viruses, antigens packaged in recombinant viral vectors and naked DNA 19. Any of the above may be able, under appropriate conditions, to elicit dominant CI, HI or both, depending on the adjuvant, antigen dose, route of immunization and cytokine profile of the vaccinee.

Consequences of simultaneous strong CI and HI Although optimal vaccine strategy may dictate that

we strive for maximum simultaneous CI and HI, the tendency of the immune system to self-regulate by reducing one arm while increasing the other may be providing a subtle but important message. Would maxi- mizing both the cellular and humoral components of immunity initiate a runaway immune system that would result in autoimmune diseases? There is little doubt that we can and eventually will achieve the 'most of both worlds', through genetically engineered vaccines and adjuvants, cytokine-redirected immune responses and D N A immunization. We should, how- ever, be aware that the 'most of both worlds' does not necessarily translate into the 'best of both worlds', as it is likely that the natural course of an immune response has had a selective evolutionary advantage for the individual.

References 1 Clerici, M. and Shearer, G. M. (1994) hmnunol. Today 15, 575-581 2 Mosmann, T. IL. and Coffman, R. L. 0989) Annu. Rev. Immuuol. 7,

145-173 3 Komagnani, S. (1991) lmmunol. Today 12, 256-257

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4 Fiorentino, D. F., Bond, M. W. and Mosmann, T. R. (1989)2". Exp. Med. 170, 2081-2098

5 "['rinchiere, G. (1993) lmmunoL Today 14, 335-338 6 ltilleman, M. (1994)AIDS Res. Hum. Retroviruses 10, 1409-1419 7 Koup, R. A. and Ho, D. D. (1994) Nature 370, 416 8 Liew, F. Y. and Parish, C. R. (1972) Cell. lmmunol. 4, 66-85 9 {]lerici, M. et al. (1994) AIDS 8, 1391-1395

10 <]lerici, M. et aL (1991) Eur.J. 1mmunol. 21, 1345-1349 11 Kovacs, J. A. et aL (1993)J. Clin. Invest. 92, 919 928 12 Salk, J., Bretscher, P. A., Salk, P. L., Clerici, M. and Shearer, G. M.

(1993) Science 260, 1270-1272 13 Shearer, G. M. and Clerici, M. (1996) bnmunol. Today 17, 21-24 14 Bretscher, P. A. (1992) Immunol. Today 13, 342-345 15 Bretscher, P. A. (1994) lmmunobiology 4, 548-554 16 Zhou, F. and Huang, L. (1993) Vaccine 11, 1139-1144 17 Thompson, C. B. (1995) Cell 81,979-982 18 Clerici, M., DePahna, L., Roilides, E., Baker, R. and Shearer, G. M.

(1993)J. Clin. Invest. 91, 2829-2836 19 McDonnel], W. M. and Askari, F. K. (1995) New Engl.J. Med. 334,

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Integrated approaches to the design of media and feeding

strategies for fed-batch cultures of animal cells

Liangzhi Xie and Daniel I. C. V,

Animal cell culture has become an important approach for the production of

biologically functional proteins for human therapy. The quantity and quality of protein

production are influenced by the culture environment, which is subject to change

over the course of cell cultivation. Therefore, it is vital to design an optimal culture

environment and control it within an optimal region to maximize the productivity.

This requires that the factors affecting the culture environment (nutrient

concentrations, by-product accumulation, pH and osmolality) and cell growth be

integrated into the design of culture media for fed-batch animal cell cultures.

The capability to secrete properly folded proteins with post-translational modifications, such as glycosylation, makes animal cells the ideal hosts for the production of various proteins with biological functions for diag- no.,is and therapy. In industry, animal cell cultivation has been practised most often in a batch mode. How- ever, many other operating modes, such as fed-batch, continuous and perfusion, have been extensively stud- ied. Simplicity in process control and operation, result- ing in process consistency, low contamination rate and low operating costs are some of the advantages of batch culture; the disadvantages are low product titer and pro ductivity, which are two important parameters that on,:." wants to maximize. The desired product is

L. )'re is at the Merck Research Laborator),, Merck Co., Inc., I~'P 26- 145, PO Box 4, Sumne),tow, Pike, West Point, PA 19486, USA. D. [. C. Wang (dicwang@mit.edu) is at the Biotechnology Process Engineering Center, Department ql" Chemical Engineering, Massachusetts Institute of Technology, Cambridge, :~,Lq 02 I39, USA.

secreted by viable cells, hence product concentration is directly proportional to the integrated viable cell concentration with time. Obviously, a high viable cell density, maintained for a long time is required to maximize the product concentration. However, this mainly depends upon the composition of the medium employed. It is well known that animal ceils require many essential nutrients to survive in vitro. These include glucose, essential amino acids, vitamins, serum components and inorganic salts. Cell growth not only consumes nutrients but also generates by-products such as ammonia and lactate, which are known to be toxic to animal cells, and NAA (see Glossary). The accumulation of NAAs may alter the medium osmo- lality. In a batch culture, the theoretical maximum cell density, is limited by the amount of nutrients present in the initial medium. Therefore, the nutrient level in the culture medium needs to be increased. However, the actual maximum cell density, depends on many other parameters. The depletion of key nutrients,

Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0167 - 7799/97/$17.00. PII: S0167-7799(97)01014-7 TIBTECH MARCH 1997 (VOL 15)